Compounds, composition, methods, targets for cancer therapy

ABSTRACT

This invention points to biological molecules and/or sequences that can be used as target for designing drug for treating cancer. This invention also points to biological sequences that can be used to target those molecules as a mean to treat cancer.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No. 14/151,539, filed Jan. 9, 2014, which is a continuation of U.S. application Ser. No. 12/667,687, filed on Jan. 4, 2010 (now U.S. Pat. No. 8,680,129), which is a Continuation in Part of PCT/US2008/069106 filed on Jul. 2,2008 which claims priority from U.S. Provisional Application Nos. 60/929,535, filed Jul. 2, 2007 and 61/156,507, filed Mar. 1, 2009, which are herein incorporated by reference in their entirety.

GOVERNMENT INTERESTS

The research carried out in the present application was supported in part by NIH. The government may have certain rights in the invention of the present application.

SEQUENCE LISTING

Attached .txt file entitled: PCT/US2008/069106-SequenceListing: The sequence Listing contains the following sequences: Nucleotide sequence of pUC19 and pCDNA3; Nucleotide sequences: Transcripts of TRPC6 (NM_004621), SH3PXD2B (NM_001017995), MAGI3 (NM_152900), TMEM182 (NM_144632), C6orf108(NM_199184); Peptide sequences: TRPC6 (NP_004612), SH3PXD2B (NP_001017995), MAGI-3 (NP_690864), TMEM182 (NP_653233), C6orf108 (NP_954653); Double strand RNA sequences: siRNA1, siRNA 2, and siRNA3.

DESCRIPTION OF THE INVENTION

Technical Field of the Invention

This invention relates to the fields of oncology and chemotherapy. Specifically, the invention provides novel compounds, methods, pharmaceutical composition and targets for more efficient and less or non cytotoxic treatments of cancer.

Background Art of the Invention

Up to date, chemotherapy and radiation therapy are still the mainstream for cancer treatment. These treatments were based on targeting proliferating cells rather than cancer cells only, which is also the cause fundamental basis of lethal side effects from these treatments. Targeted therapy, a new generation of cancer treatment is aimed to target cancer specific changes of molecules and signaling pathways to induce cancer cell death, but limit such effects on normal cells. Enormous efforts have been made in finding the targets and the ways of targeting the targets inside the cells as a treatment. However, up to date, the success rate of this new generation is limited. One major challenge comes from the complexity of cellular regulation mechanisms and overlapping pathways inside the cells.

Aberrant Nuclear factor-kappa B (NF-κB) activation has been associated with a variety of tumors and cancer cells for oncogenesis, regulation of cell proliferation, inhibition of apoptosis, promoting angiogenesis, tumor invasion and metastasis as well as cancer cell resistance to chemotherapy and radiation therapy treatments (Kim H J. Hawke N. and Baldwin A S, NF-κB and IKK as therapeutic targets in cancer, Cell Death ad Differentiation, (2006) 13:738-47; Karin M, Nuclear factor-κB in cancer development and progression, (2006) Natur 441:431-6). Inhibition NF-κB activity could facilitate cancer cell death and sensitize cancer cells to chemotherapy drugs and radiation therapy (Kim H J, Hawke N, and Baldwin A S, NF-κB and IKK as therapeutic targets in cancer, Cell Death and Differentiation, (2006) 13:738-47; Karin M. Nuclear factor-κB in cancer development and progression, (2006) Natur 441:431-6; Chikashi Nakanishi and Masakazu Toi. Nuclear Factor-κB Inhibitors As Sensitizers To Anticancer Drugs. NATURE REVIEWS CANCER (2005) 5:297-309).

There are two similar, but different IκB Kinases (IKK1 and IKK2) that are up stream regulator of NF-κB activity. In addition, alternative NF-κB activation pathways, such as protein kinase CK2 (CK2), also exist. In most cancer cells, NF-κB is constitutively activated. In addition to the IKK classic pathway, these alternative NF-κB activation pathways may also contribute to the aberrant NF-κB activity in cancer cells (Ming Yu. Jason Yeh, and Carter Van Waes Protein Kinase CK2 Mediates Inhibitor-Kappa B Kinase and Aberrant Nuclear Factor-κB Activation by Serum Factor(s) in Head and Neck Squamous Carcinoma Cells Cancer Research, 2006 Jul. 1; 66(13): 6722-6731 and other for NFKB activation). Dozens of IKK inhibitors have been produced and are in trials for treating anti-inflammatory diseases. However, for treating cancer with these IKK inhibitors, these efforts have yielded results far from spectacular (Chikashi Nakanishi and Masakazu Toi, Nuclear Factor-κB Inhibitors As Sensitizers To Anticancer Drugs. NATURE REVIEWS CANCER (2005) 5:297-309).

More recent studies pointed to the balance between NF-κB activity and C-Jun-N-Terminal-kinase (JNK) activity, which regulates cell death or proliferation (reviews). In this theory, NF-κB and JNK cross talk through reactive oxygen species (ROS). Both JNK and NF-κB activity leads to Cell proliferation. However, ROS induces prolonged JNK activation that will induce programmed cell death. Conversely, activated NF-κB suppresses ROS and, hence, suppress ROS induced prolonged JNK activation. Therefore, inhibiting NF-κB while activating JNK would switche the balance to programmed cell death. However, up to date, no such treatment method has been reported. Most importantly, although this specific theory has been proposed, none has prior succeeded in demonstrating this effect.

ROS are potentially harmful by-products of normal cellular metabolism that directly affect cellular functions. ROS are also acts messenger and indispensable for signal transduction pathways that regulate cell growth and reduction-oxidation (redox) status. However, overproduction of these highly reactive oxygen metabolites can initiate lethal chain reactions, which involve oxidation and damage to structures that are crucial for cellular integrity and survival. In fact, many antitumor agents, such as vinblastine, cisplatin, mitomycin C, doxorubicin, camptothecin, inostamycin, neocarzinostatin and many others exhibit antitumor activity via ROS-dependent activation of apoptotic cell death, suggesting potential use of ROS as a fundamental antitumor principle. The “oxidation therapy” a unique anticancer strategy by inducing the generation of ROS directly to solid tumors as cytotoxic oxystress for cancer treatment has been developed. However no successful and practical results were obtained probably because of the lack of tumor selective ROS delivery and hence resulting in subsequent induction of severe side effects (Fang, J., Nakamura, H., and Iyer, A. K. Tumor-targeted induction of oxystress for cancer therapy. J Drug Target. 15: 475-486, 2007).

One of the unique features of cancer cells is their dependency on aerobic glycolysis, the “Warburg effect” that most cancer cells predominantly produce energy by glycolysis followed by lactic acid fermentation in the cytosol, rather than oxidation of pyruvate in mitochondria by most normal cells (Warburg O., Science 123:309, 1956). Along with this Aerobic glycolysis is that cancer cells consume oxygen through trans-plasma membrane electron transport (tPMET) at cell surface that oxidizes the NADH⁺ that generated from the glycolysis processes in cytosol and to generate ATP (Heart, P M, Curr Mol Med, 2006, 6:895). The tPMET is mediated by NADH Oxidases (NOX) located on cell plasma membrane. This process oxidizes intracellular NADH and recycles it to maintain the intracellular NADH/NAD+ ratio to support glycolytic ATP. As ATP production contributes substantially to fulfilling the energy requirements of rapidly dividing cells, such as cancer cells, and that tPMET is the major source for cancer cell energy production that is different from normal cells, which perform energy metabolism and consume oxygen in mitochondrial. Therefore, targeting tPMET could be a strategy for cancer specific treatment. This concept was initially proposed by Herst P M, and Berridge M V based on the facts that the compounds that affect tPMET also affect cancer cell survival (Herst P M, Berridge M V, Curr Mol Med 6:895, 2006). It was further hypothesed that blocking the electron transport through interfering with membrane ubiquitou recycling, destabilizing the redox status of the cell membrane that may stimulate acid sphingo-myelinase activity, result in the conversion of sphigomyelin to ceramide that will lead to formation of ceramide-enriched membrane islands, which lead to apoptosis (Dumitru, C. A. et al, 2006, Oncogene 25:5612-25). Based on this hypothesis, Berridge et al proposed to make drugs specifically located to the plasma membrane without entering the cell as a novel anticancer drug development strategy. However, up to the date of filing this application, no such development had been reported.

Hypoxia inducing factor (HIF) and pyruvate kinase 2 (PK-M2) are known to be responsible to the switch to aerobic glycolysis by cancer cells, but targeting PK-M2 resulted intolerable side effects. High HIF expression levels and activities have been associated with all cancer cells that make cancer cells resistant to low oxygen levels. Furthermore, cancer cells are actively undergoing catabolism, which result high demands for reducing sources that oxidize the NADH+ generated from the glycolysis process, which further makes cancer cells can survival in close to zero oxygen levels. Single inhibition of HIF seems not sufficient to kill cancer cells. More effective inhibition of cancer cell specific respiration is still lacking and has been sought hardly.

Apigenin is a naturally occurring plant flavone (4′,5,7,-trihydroxyflavone) abundantly present in common fruits and vegetables including apple, parsley, onions, oranges, tea, chamomile, wheat sprouts and some seasonings. Apigenin is a multi function signal conduction agent and has been shown to possess remarkable anti-inflammatory, antioxidant and anti-carcinogenic properties and is currently under active study. Studies on the biological effects of apigenin at cellular and molecular levels have found that apigenin interferes with a wide range of critical molecules and signaling and regulatory processes in the cells, including depleting the HER2 protein and suppressing the Her2/Her3-phosphatidylinositide 3-kinase/AKT pathway (Way, T. D. and Lin, J. K. Role of HER2/HERS co-receptor in breast carcinogenesis. Future Oncol, 1: 841-849, 2005), inhibit HIF, PKC, CDK, VEGF NF-□B, CK2, AKT, MAPK, AR and BR pathways, activate wild type p53, modulate the deregulated cell cycle checkpoint and induce apoptosis (Induction of caspase-dependent, p53-mediated apoptosis by apigenin in human neuroblastoma—Torkin et al. 4 (1): 1— . . . 2007; Apigenin Inhibits Expression of Vascular Endothelial Growth Factor and Angiogenesis in Human Lung Cancer Cells: Implication of . . . 2007; Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70S6K1 and HDM2/p53 pathways—Fang et al. 19 (3): 342—The FASEB . . . 2007; Balasubramanian. S. and Eckert, R. L. Keratinocyte proliferation, differentiation, and apoptosis—differential mechanisms of regulation by curcumin, EGCG and apigenin. Toxicol Appl Pharmacol, 224: 214-219, 2007; Dirt. D. F., Walker, B., Tihbels, M. G.., and Bresnick, E. Anti-mutagenesis and anti-promotion by apigenin, robinetin and indole-3-carbinol. Carcinogenesis, 7: 959-963, 1986; Patel, D., Shukla, S., and Gupta, S. Apigenin and cancer chemoprevention: progress, potential and promise (review). Int J Oncol 30:233-245, 2007; Sato, F., Matsukawa, Y, Malsumoto, K., Nishino, H., and Sakai, T. Apigenin induces morphological differentiation and G2-M arrest in rat neuronal cells. Biochem Biophys Res Commun 1994 Oct. 28; 204: 578-584, 1994). In addition, apigenin has also been reported lo generate ROS, which disrupt mitochondrial membranes. Current research trials indicate that it may reduce DNA oxidative damage; inhibit the growth of human leukemia cells and induced these cells to differentiate; inhibit cancer cell signal transduction and induce apoptosis; act as an anti-inflammatory; and as an anti-spasmodic or spasmolytic. More than 100 patent applications related to apigenin have been filed. Among those, apigenin was claimed to be used as a drug for treating inflammatory and autoimmune diseases. In addition, apigenin was also claimed for the use as a cancer chemoprevention drug and as adjunct use for enhancing the effects of chemotherapy drugs for cancer treatment at 10 μM concentration (US Patent Application 20060189680). However, as a chemo sensitizer, the efficacy effect of apigenin is limited. To be a cytotoxic drug for treating cancer, apigenin has to be combined with other treatments. Other isoforms of apigenin, other flavonoids, isoflavonoids including, naturally existed, modified or synthetic including phenoxodiol a synthetic isoflevene, have also been found with similar function of apigenin. All of those need to be combined with chemotherapy drugs for cancer treatment.

SUMMARY OF THE INVENTION

A more efficient and cancer specific anticancer treatment can be achieved by combining inhibition of cancer cell surface respiration with inhibiting its hypoxia response.

The present invention provides pharmaceutical composition and combinational composition and methods for the treatments of cancer and genes as drug targets to enable the treatment of cancer in a mammal to synergize cancer specific cell death with less or no cytotoxic side effects including:

A pharmaceutical composition and a method for treating cancer by targeting the tPMET of cancer cells lo block the tarns plasma membrane electron transfer and/or uncoupling the oxidative phosphorylation across the cell plasma membrane without affecting the same function at the mitochondria membrane in combination inhibition of cellular responses to hypoxia to reach a synergistic therapeutic effect of inducing cancer specific cell death for cancer treatment;

A compound and its required chemical structure for targeting tPMET for cancer treatment;

A use of WST-3 and any of the valid substitutes that are capable of blocking the tPMET by uncoupling the oxidative phposphorylation on cell plasma membrane for the said combination treatment;

A pharmaceutical composition and a method for treating cancer by combining WST-3 or its valid substitutes with apiginin or its valid substitutes as an cancer specific and less toxic anticancer treatment;

A use of a reagent WST-1r comprising water soluble tetrozolium salts and intermediate electron acceptors as a drug to interfere tPMET for the said combination treatment;

A pharmaceutical composition of WST-1r and any of the valid substitutes of WST-1r for the said combination treatment that are capable of conducting trans-plasma membrane electron transport and induces ROS. The WST-1r and any of the valid substitutes of WST-1 is a mixture of tetrazolium salt and an electron coupling reagent (IEA), or at least one of the tetrozolium salt or at least one of the IEA in optimized concentration. The compounds may be administered in a pharmaceutically acceptable carrier medium.

A pharmaceutical composition and a method for treating cancer by combining WST-1r or its valid substitutes with apiginin or its valid substitutes as an cancer specific and less toxic anticancer treatment;

Selected genes, molecules, and polynucleotide sequences and polypeptide sequences are provided as target for designing drugs for the treatment of a cancer in a patient in need. These targets are the human transcripts, and their corresponding protein/peptide molecules and/or the genomic DNA sequences that are selected front the blast analysis of the DNA sequence of pUC19 DNA vector against human genome and transcripts, the DNA sequences of which mapped to the human transcripts and/or genomic sequences in short pieces. The transcripts and their corresponding coding molecules are targets for enhancing the efficacy of the treatments of cancer. Other sequences that, thus, mapped to human genomic sequences may be used as targets as well as being used for targeting these corresponding genes. The potential drugs that can be designed to targeting these targets include, but not limited to, siRNA, small molecule inhibitors, peptides inhibitors, anti-sense RNA, anti-sense Oligo, antibodies, antibody fragments, proteins, dominant negative DNA vectors and Interferon (IFN). In a particular embodiment of this invention, these targets are, but not limited to, polynucleotide sequences of TRPC6 (SEQ ID NO: 2), MAGI-3 (SEQ ID NO: 4), TMEM182 (SEQ ID NO: 5), SH3PXD2B (SEQ ID NO: 3), or c60rf108 (SEQ ID NO: 14), and the polypeptide sequences of TRPC6 (SEQ ID NO: 6), MAGI-3 (SEQ ID NO: 8), TMEM182 (SEQ ID NO: 9), SH3PXD2B (SEQ ID NO: 7), or c60rf108 (SEQ ID NO: 15). The sequence to target human genomic sequence and or transcripts are, but not limited to, puc19 DNA vector (SEQ ID NO: 1), pc DNA3 vector (SEQ ID NO: 13), siRNA2 (SEQ ID NO: 10-12). Synthetic siRNA that against these target genes (SEQ ID NO: 2-5 and SEQ ID NO: 14) were selected for demonstrating the potential use of these genes as a target for the combination treatment for cancer.

A method for treating a cancer in a patient in need thereof comprising administering to the patient, concurrently or sequentially, a therapeutically effective amount of (1) at least one of the transfection of puc19 DNA vector or administering at least one of the substitutes of puc19 DNA transfection and (2) at least one IKK inhibitor and (3) an additional third agent, WST-1r or at least one of the valid substitutes of WST-1r, in a pharmaceutically acceptable carrier medium. Wherein said combination enhances the induction of cancer cell death while otherwise any of these agents separately are demonstrated not to be toxic.

The valid substitutes for Puc19 DNA transfection are selected from the group consisting of (1) type 1 IFN, (2) Synthetic small interfering RNAs (siRNA) the nucleotide sequence SEQ ID NO 10-12 of which mapped to both the DNA sequence of the pUC19 DNA vector and human transcripts and genome DNA sequences, (3) the biological compounds selected from the group consisting of biological and non-biological organic or non-organic compounds. The said method of screening compounds, wherein said biological chemicals are further selected from the group of polypeptides, proteins, peptides, antibodies, antibody fragments, nucleic acids, and polynucleotide the products of which interact and interfere said selected targets of the polynucleotide sequences of TRPC6 (SEQ ID NO: 2), MAGI-3 (SEQ ID NO: 4), TMEM182 (SEQ ID NO: 5), SH3PXD2B (SEQ ID NO: 3), or c60rf108 (SEQ ID NO: 14), and the polypeptide sequences of TRPC6 (SEQ ID NO: 6), MAGI-3 (SEQ ID NO: 8), TMEM 182 (SEQ ID NO: 9), SH3PXD2B (SEQ ID NO: 7), or c60rf108 (SEQ ID NO: 15). Synthetic siRNA that against these target genes (SEQ ID NO: 2 to 5 and SEQ ID NO: 14) were selected for demonstrating the potential use of these genes as a target for the combination treatment for cancer.

A method of inducing programmed cell death of cancer cells in a malignant cell population, and treating a patient with cancer comprising the use of a combination therapy. The combination therapy of the present invention comprises administering an effective dose of WST-1r or any valid substitutes, that is capable of conducting trans-plasma membrane electron transfer and induces ROS in a cell and apigenin, a multi-function inhibitor that inhibits HIF, CK2, NF-κB activity and other molecules and/or signaling pathways, or at least one of the IKK inhibitor. The said combination treatment enhances apigenin anti-neoplasm effect and synergizes the induced cancer cell death;

A method is provided for treating a cancer in a patient in need thereof by administering to the patient, concurrently or sequentially, a therapeutically effective amount of at least one GSK3β inhibitor and protein kinase CK2 (CK2) inhibitor and addition of a third agent, WST-1r. In a particular embodiment of the invention, the preferred at least one GSK30 inhibitor is LiCl and the preferred protein kinase CK2 (CK2) inhibitor is Apigenin. The compounds may be administered in a pharmaceutically acceptable carrier medium. Wherein said combination enhances the induction of cancer cell death otherwise any of these agents separately are demonstrated not to be toxic;

A method is provided for treating cancer in a patient in need comprising administering, concurrently or sequentially, a therapeutically effective amount of a combination of a selective Puc19 DNA trasnfection or at least one of any of the valid substitutes of puc19 transfection as listed above in combination with at least one of a selected approved chemotherapeutic agents. Wherein said Puc19 DNA trasnfection or administering at least one of the valid substitutes of puc19 transfection being capable of substantially enhancing anti-neoplastic effects of said proved chemotherapeutic agents, substantially reducing toxic side effects of said chemotherapeutic agents, or a combination thereof, wherein said Puc19 DNA trasnfection or at least one of the valid substitutes has a substantial effect on activity of said chemotherapeutic agents;

A method is provided for synergistically inhibiting NF-□BNF-KAPPAB activity in cancer cells and in a patient in need thereof by administering to the cells or patient, concurrently or sequentially, a therapeutically effective amount of at least one Dominant negative kinase dead IKKI DNA vector (IKK1-KA) and at least one Dominant negative kinase dead IKK2 DNA vector (IKK2-KA). The at least one Dominant negative kinase dead IKK1-KA or IKK2-KA may be substituted by IKK inhibitors selected from the group consisting of (IKK inhibitor list). The compounds may be administered in a pharmaceutically acceptable carrier medium. This combinational inhibition effect may be further enhanced by adding a third agent, WST-1r or the valid substitutes of WST-1r, for further induction of cancer cell death;

A method of inducing cancer cell death, and treating a patient comprising the use of a combination therapy. The combination therapy of the present invention comprises administering an effective dose of at least a compound that inhibits NF-κB activity and at least one compound that inhibits STAT3 in a preferred embodiment, the compound that inhibits NF-κB activity is apigenin or an IKK inhibitor or a CK2 inhibitor and the compound that inhibits STAT is stattic. The compounds may be administered in a pharmaceutically acceptable carrier;

A use of the combination therapy to treat cancers comprising administering IKK inhibitor or apigenin and a STAT3 inhibitor, stattic. In one preferred embodiment, the cancers are selected from the group consisting of a subtype of head and neck squamous carcinoma.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Chemical Structure of DNP and WST-3

FIG. 2. Chart of endogenous NF-κB down stream gene expression levels.

FIG. 3. Chart of cell survival from IKK-KA transfection in combination with or without WST-1 treatment.

FIG. 4. Chart of WST-1 promotes HT1080 Human Sarcoma cell death by triple combinatination Treatment

FIG. 5. Chart of combination of LiCl and Apigenin and WST-1r treatment.

FIG. 6. Chart of pUC19 DNA transfection synergize chemotherapeutic drug effect

FIG. 7. Chart of Time Course of ROS generation after combination treatment of WST-1r, CCK8 with apigenin and IKK inhibitor III

FIG. 8. Chart of combination treatment with apigenin and WST-1r synergizes induced cancer cell death.

FIG. 9. Chart for Differential Responses to WST-1r and Apigenin Combination Treatment from Human Non-Cancer Cells and Human Head and Neck Cancer Cells.

FIG. 10. Chart of Time course and Dose Response of WST-1r and Dose-Response of apigenin involved in the combination treatment of WST-1r with apigenin.

FIG. 11. Chart showing Effect of Combination treatment with IKK Inhibitor and WST-1r on melanoma cell lines

FIG. 12. Chart of effects of treatment order of WST-1r and BMS345541 on induced cell death

FIG. 13. Chart showing WST-1r and Apigenin combination treatment induced JNK Phosphrylation

FIG. 14. Chart of Effects of CCK8 and XTTas substitute of WST-1r in combination treatment with apigenin for inducing cancer cell death

FIG. 15. Chart of Combination Treatment of Apigenin with WST derivatives, mPMS or Combination of WST and mPMS

FIG. 16. Chart of Dose-Response of Apigenin and mPMS Combination Treatment

FIG. 17. Chart of Differential cellular responses to mPMS treatment

FIG. 18. Chart of Effect of Combination WST-3 with Apigenin On Cell Death

FIG. 19. Chart of Effect of WST-3, and WST-3+mPMS in Combination with Apigenin On Inducing Cancer Cell Death

FIG. 20. Chart of siRNA substitution of pUC19 for enhancing WST-1r-IKK inhibitor combination Treatment Effect

FIG. 21. Chart for Effect of Type I INF Substitute pUC19 for combination Cancer treatment

FIG. 22. Chart of enhancement of Taxel Efficacy Effects by Combination of Puc19 DNA sequence derived siRNA with Taxel

FIG. 23. Immage of Induction od ROS Generation by WST-1r and the combination treatment.

FIG. 24. Chart of Dose Response of ROS generation after combination treatment of WST-1r, CCK8 with apigenin and IKK inhibitor III

DETAILED DESCRIPTION OF THE INVENTION I. General Description

The efficacy of anticancer therapy can be enhanced by combination of proper selected compounds, biological molecules and drugs that block the tPMET and cell surface respiration in combination with inhibiting cellular hypoxia responses to induce synergistic and cancer cell specific cell death. Several combinational pharmaceutical compositions and methods for anticancer treatment are described. The development of these pharmaceutical compositions were based on the following discoveries:

The discovery the use of WST-3, WST-1r and their valid substitutes as drugs for combination therapies by combining with apigenin, IKK inhibitors or Puc19 DNA or any of its valid substitutes that induced synergetic and cancer specific cell death,

The discovery the structure and function features of WST-3 representing a class chemicals of a cell surface oxidative-phosphorylation uncpupler and the corresponding principle to design a chemical compound for targeting tPMET and blocking cell surface oxidative-phosphorylaition, tPMET and cell respiration for the said combination anticancer treatment

(3) The use of puc19 DNA sequences and the corresponding siRNAs as anti cancer drugs as well as the discovery of the corresponding genes as target for developing anticancer therapy.

(4) The discovery of the method and combinational composition of WST-3 and apigenin or their valid substitutes as anticancer treatment.

(5) The discovery of the method and combinational composition of WST-1r and apigenin or their valid substitutes as anticancer treatment.

The first and the second discoveries led to the identification of classes of chemicals and corresponding pharmaceutical compositions of using these chemical compounds as drugs for the combination treatment for cancer. The third finding further led to the discovery of several genes as targets for developing anticancer drugs. These are rarely studied genes and some of them are still in hypothetical gene status. Together, these findings led to establishing several combinational treatment methods for cancer therapy.

In one embodiment, the pharmaceutical composition of WST-3 was described as a cell surface oxidative-phosphorylation uncouple for the use as drugs for combinational treatment of cancer.

In one embodiment, the pharmaceutical composition of WST-1r was described an agent that interferes tPMET for the use as drugs for combinational treatment of cancer.

Yet in another embodiment, the classes of chemical compounds of WST-3 and their special chemical structures for designing drugs to direct target the tPMET and as an uncoupler to block the cell surface energy metabolism and cell surface respiration are descried for the use of the combination treatment for cancer.

Yet in another embodiment, the classes of chemical compounds and the combination of these compounds that can form the formula of WST-1r and the valid substitutes of WST-1r are descried for the use of the combination treatment for cancer.

In one of the embodiments, Puc19 DNA vector was found to have biological effect on mammalian and human cancer cells and was used as a drug for combination treatment with WST-1r reagents and with or without IKK inhibitors.

In another embodiment, Puc19 DNA vector was used as a drug in combination with chemotherapeutic drug for enhancing the therapeutic effect of these chemotherapeutic drugs for the treatment of cancer.

According to the above embodiments, small interfering RNAs (SEQ-1D No: 10-12), the sequence of which were derived from the nucleotide sequence of Puc19 DNA vector, were described for the use of combination treatments for cancer.

Yet also according to the above embodiments, human genes (TRPC6 (SEQ ID NO: 2), MAGI-3 (SEQ ID NO: 4), TMEM 182 (SEQ ID NO: 5), SH3PXD2B (SEQ ID NO: 3), or c60rf108 (SEQ ID NO: 14), and the polypeptide sequences of TRPC6 (SEQ ID NO: 6). MAGI-3(SEQ ID NO: 8), TMEM182 (SEQ ID NO: 9), SH3PXD2B (SEQ ID NO: 7), or c60rf108 (SEQ ID NO: 15)) that were selected bused on Puc19 DNA sequence analysis and the biological function of the corresponding siRNAs to be used as target for drug development for the treatment of cancer are described.

Yet another embodiment, wherein said the valid substitutes of Puc19 DNA that were selected from biological and non-biological compounds and their effects in combination with WST-1r or the valid substitutes of WST-1r and with or without IKK inhibitor is described. Wherein said the biological compound for the valid substitutes of Puc19 DNA include different members of Interferon and all the siRNAs mentioned above.

Yet another embodiment, wherein said the valid substitutes of Puc19 DNA that were selected from biological and non-biological compounds and their effects in combination with chemotherapeutic drugs is described. Wherein said the biological compound for the valid substitutes of Puc19 DNA include different members of Interferon and all the siRNAs mentioned above.

In another embodiment, a medical use of combination treatment for cancer comprising apigenin, the flavonoids or at least one IKK inhibitor and WST-1r is described.

Yet in another embodiment, a medical use of combination treatment for cancer comprising at least one Protein kinase II (CK2) inhibitor, apigenin, at least one GSK3□□inhibitor, Lithium chloride, and WST-1r for enhancing treatment effect is described.

II. Definitions

The term “pUC19 DNA” is a DNA cloning vector (SEQ ID #1) that amplifies in prokaryotic cells. DNA sequence of this vector was originally submitted to NCBI gene bank by J. Messing, Waksman Institute, NJ on 3 Mar. 1986 and revised by F. Pfeiffer on 16 Dec. 1986. In the present description, pUC19 has been used as a drug for anticancer therapy by transfected into human cancer cells.

The data suggests that the DNA sequence that composes this DNA vector has biological effects in cultured human cancer cells that lead to synergistic cell death when combined with other treatments to these cells as described in this description. Blast analysis of the DNA sequence of pUC19 against human genome and transcripts for short matches showed multiple short sequences aligned to varies locations of flanking sequences of human genome and transcripts (Blast result is attached to this application). In the present description, pUC19 represents the combination of short DNA sequences, usually 15-100 bases that mapped to human transcripts and/or flanking regions of genes of human genome DNA sequences. Accordingly, the corresponding gene products are the targets of the pUC19. The polynucleotide sequences and amino acid sequences that include but not limited to siRNA, miRNA, shRNA, peptide that are directly derived from the pUC19 DNA sequence as well as derived from the corresponding genes and small molecules and antibodies that can interact and/or inhibit the function and activity of these corresponding molecules as direct gene products of their DNA sequences, the DNA sequences of their corresponding gene contain these short matched DNA sequences from the DNA sequence of pUC19. The matched DNA sequences don't have to be exact matches. The matched DNA sequences can vary slightly, 10%, 20%, and even up to 30-40%.

The term “pcDNA3m DNA” is a mammalian expression vector version 3.1 with modifications [SEQ ID #13]. DNA sequence of this vector was originally derived from the pUC19 with further modifications and obtained from Invitrogen, which has discontinued the production and selling of this vector. pcDNA3 has been transacted into human cancer cells by chemical or liposome based DNA transfection reagents. Similar to pUC19, the DNA sequence that composes this DNA vector have biological effects in cultured human cancer cells that lead to synergistic cell death when combined with other treatments to these cells as described. In the present description, pcDNA3 represents the short DNA sequences, usually 15-100 bases that mapped to human transcripts and/or human genome DNA sequences, and their corresponding gene products that include but not limited to siRNA, miRNA, shRNA, peptide that are directly derived from the DNA sequence of this vector and small molecules that can interact and/or inhibit the function and activity of these corresponding molecules as direct gene products of their gene sequences, the DNA sequences of their corresponding gene contain these short matched DNA sequences from the DNA sequence of pcDNA3. The matched DNA sequences don't have to be exact matches. The matched DNA sequences can vary up to 30-40% changes.

The term “siRNA1” [SEQ ID #10] is a siRNA designed based on and derived from the DNA sequence of pUC19 [SEQ ID #1]. This siRNA sequence matches to the human transcript of Homo sapiens transient receptor potential cation channel, subfamily C, member 6 (TRPC6, GeneID: 7225), mRNA (gi|19923256|NM_004621.3) synonyms: TRP6, FSGS2, FLJ11098. In the present description, siRNA1 was used as a drug For targeting TRPC6 for the treatment of cancer. As in general the siRNA sequence can vary slightly, 10%, 20% and even 30-40% of the exact sequence of the transcript.

The term “siRNA3” [SEQ ID #12], is a siRNA designed based on and derived from the pUC19DNA sequence [SEQ ID #1]. This siRNA sequence matches to the human transcript of Homo sapiens membrane associated guanylate kinase, WW and PDZ domain containing 3 (MAGI3, GeneID: 260425), transcript variant 2, mRNA (NM_152900.1) synonyms: MAGI-3, MGC163281 and the Homo sapiens transmembrane protein 182 (TMEM182, GeneID: 130827), mRNA (NM_144632.2). In the present description, siRNA3 was used as drug for targeting MAGI3 and/or TMEM182 for the treatment of cancer. As in general the siRNA sequence can vary slightly, 10%, 20% and even 30-40% of the exact sequence of the transcript.

The term “siRNA2” [SEQ ID #11] is a siRNA designed based on and derived from the DNA sequence of pUC19 [SEQ ID#1]. The 2/3 of this siRNA sequence matches to the human transcript of Homo sapiens SH3 and PX domains 2B (SH3PXD2B), mRNA. (SH3PXD2B, GeneID: 285590), mRNA (NM_001017995) synonyms: HOFI; FLJ20831; KIAA1295. In addition, this sequence also mapped to more than 45 sites within flankin sequences of human genome. In the present description, siRNA2 was used for targeting SH3PXD2B and all the other potential DNA sequences in the human genome for the treatment of cancer. As in general the siRNA sequence can vary 30-40% of the exact sequence of the transcript.

The term “TRPC6” [Nucleotide SEQ ID #2. Peptide SEQ ID #6] represents human transcript of Homo sapiens transient receptor potential cation channel, subfamily C, member 6 (TRPC6, GeneID: 7225), mRNA (NM_004621.3) synonyms: TRPC6, FSGS2, FLJ11098. In the present description, TRPC6 is a target for developing anticancer treatment, TRPC6 can be targeted by any means that alter its expression levels and activities at functioning levels including but not limited to poly nucleotides, such as siRNA, shRNA, anti-sense RNA, anti-sense DNA oligo, and dominant negative DNA vectors, peptide and amino acid sequences, such as peptide, and antibodies, and small molecule inhibitors. The TRPC6 has been previous reported as a potential target for cancer treatment, but no report regarding the use of TRPC6 as a target for a combinational cancer treatment with IKK inhibitors, WST1r or chemotherapy drugs to reach the synergistic effect of promoting cancer cell death. The siRNA1 sequence described above is the preferred sequence, but this does not limit from other siRNA sequences and other means as described in this paragraph. As in general the siRNA sequence can vary slightly, 10%, 20% and even 30-40% from the exact sequence of the transcript.

The term “MAGI3” [Nucleotide SEQ ID #4, Peptide SEQ ID #8] represents human transcript of Homo sapiens membrane associated guanylate kinase, WW and PDZ domain containing 3 (MAGI-3, GeneID: 260425), transcript variant 2, mRNA (NM_152900.1). Synonyms: MAGI-3, MGC163281. MAGI-3 is localized with ZO-1 and cingulin at tight junctions in epithelial cells, whereas MAGI-3 was found in E-cadherin-based cell-cell contacts and in focal adhesion sites in primary cultured astrocytes (Adamsky K, Arnold K, Sabanay H, Peles E., Junctional protein MAGIKK interacts with receptor tyrosine phosphatase beta (RPTP beta) and tyrosine-phosphorylated proteins. (J Cell Sci. 2003, 116 (Pt 7): 1279-89). MAGI-3 interacts directly with LPA(2) and regulates the ability of LPA(2) to activate Erk and RhoA MAGIKK regulates LPA-induced activation of Erk and RhoA (Zhang H. Wang D, Sun H, Hall R A, Yun C Q Cell Signal. 2007 February; 19(2): 261-8. Epub 2006 Aug. 9). The function of MAGI3 has been previous linked to cancer, but no report regarding the use of MAGI3 as a target for a combinational cancer treatment with WST-1r, IKK inhibitors or chemotherapy drugs to reach the synergistic inhibition of cancer cell growth and to promote cancer cell death. In the present description, MAGI3 is a target for developing anticancer treatment. MAGI-3 can be targeted by any means that alter its expression levels and activities at functioning levels including but not limited to poly nucleotides, such as siRNA, shRNA, anti-sense RNA, anti-sense DNA oligo, and dominant negative DNA vectors, peptide and amino acid sequences, such as peptide, and antibodies, and small molecule inhibitor. The siRNA3 sequence described above is the preferred sequence, but this does not limit from other siRNA sequences and other means as described in dm paragraph. As in general the siRNA sequence can vary slightly, 10%, 20% and even 30-40% from the exact sequence of the transcript.

The term “TMEM182” [Nucleotide SEQ ID #5, Peptide SEQ ID #9] represents Homo sapiens trans-membrane protein 182 (TMEM182, GeneID: 130827), mRNA (NM_144632.2). In the present description, SH3PXD2B is a target for developing anticancer treatment. TMEM182 can be targeted by any means that alter its expression levels and activities at functioning levels including but not limited to poly nucleotides, such as siRNA, shRNA, anti-sense RNA, anti-sense DNA oligo, and dominant negative DNA vectors, peptide and amino acid sequences, such as peptide, and antibodies, and small molecule inhibitors. The siRNA3 sequence described above is the preferred sequence, but this does not limit from other siRNA sequences and other means as described in this paragraph. As in general the siRNA sequence can vary 10%, 20% and even 30-40% from the exact sequence of the transcript TMEM 182 has not been previously studied and not been linked to cancer.

The term “SH3PXD2B” [Nucleotide SEQ ID #3, Peptide SEQ ID #7] represents SH3 and PX domains 2B adaptor protein HOFI (GeneID: 285590) that contains SIB and PX domains. SH3 domains, Src homology 3 domains, bind to prolinerich ligands with moderate affinity and selectivity, preferentially to PxxP motifs; they play a role in the regulation of enzymes by intramolecular interactions, changing the subcellular localization of PX; PhoX homologous domain, present in p47phox and p40phox. Eukaryotic domain of unknown function presents in phox proteins, PLD isoforms, and a PI3K isoform. SHPXD2B has not been previously studied and not been linked to cancer. In the present description, SH3PXD2B is a target for developing anticancer treatment. SH3PXD2B can be targeted by any means that alter its expression levels and activities at functioning levels including but not limited to poly nucleotides, such as siRNA, shRNA, anti-sense RNA, anti-sense DNA oligo, and dominant negative DNA vectors, peptide and amino acid sequences, such as peptide, and antibodies, and small molecule inhibitors. The siRNA2 sequence described above is the preferred sequence, but this does not limit from other siRNA sequences and other means as described in this paragraph. As in general the siRNA sequence can vary 10%, 20% and even 30-40% from the exact sequence of the transcript.

The term “C6orf108” [Nucleotide SEQ ID #14, Peptide SEQ ID #15] represents human C6orf108 chromosome 6 open reading frame 108 [Homo sapiens] GeneID: 10591. Official Symbol C6orf108. This gene was identified on the basis of its stimulation by c-Myc protein. The exact function of this gene is not known but studies in rat suggest a role in cellular proliferation and c-Myc-mediated transformation. In the present description, C6orf108 is a target for developing anticancer treatment, C6orf108 can be targeted by any means that alter its expression levels and activities at functioning levels including but not limited to poly nucleotides, such as siRNA, shRNA, anti-sense RNA, anti-sense DNA oligo, and dominant negative DNA vectors, peptide and amino acid sequences, such as peptide, and antibodies, and small molecule inhibitors.

The term “Interferon” (IFN) is a group of cytokines produced by leucocytes and fibroblasts. The 1FN that are described herein includes all type I and type II IFNs and all the subtypes of IFN including, but not limited to IFNα A, IFNα B, IFNα C, IFNα D, IFNα F, IFNα G, IFNα H, IFNα I, IFNα J, IFNα K, IFNα 4b, IFNα WA, IFNβ, IFNγ and IL-6.

The term “WST-1c” representing a water soluble tetrazolium salt WST-1 {4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedilsulfonate} was first described by ishiyama et al in 1996 (Ishiyam M. et al Biol Pharm Bull 1996, 19:1515-20).

The term “WST-1r” represents a reagent mixture comprising WST-1c and mPMS at optimized concentration and the ratio between WST-1c and mPMS for the combination treatment. The optimized concentration and molar ration of the two components may not be the same as that of the commercial “cell proliferation kit”

The term “IEA” is the symbol of “Intermediate Electron Acceptor”.

The term “mPMS” (1-methoxy-5-methyl-phenazinium methyl sulfate) is a chemical compound acts as an “electron coupling agent/IE A” when combined with tetrazolium salts.

The term “Q1” (coenzyme Q1,2,3-Dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone) is a chemical compound act as an IEA.

The term “WST” represents the collection of a class of compounds of water soluble tetrazolium salts including, but not limited to WST-3, WST-4, WST-5. WST-8, WST-9, WST-10, WST-11, XTT, and MSN. These compounds are also impermeable to cell plasma membrane.

The term “XTT” represents a water soluble tetrazolium salt in the similar class of WST-1 as well as a reagent that composed of XTT and mPMS or coenzyme Q1.

The term “CCK8” represents a cell counting kte, which is composed of WST-8 and mPMS.

The term “valid substitutes of WST-1r” represents any compounds that can substitute the function of WST-1r, WST-1c, or the electron coupling reagent mPMS or any of the remaining components either act alone or in any type of combination among these substitutes or any type of combination with any of the component of the water soluble tetrazolium salt and IEA that comprising WST-1r to function as the WST-1r as described in this specification to reproduce the synergistic induction of cancer cell death. The term “valid substitutes of WST-1r” includes, but not limited to all the up to date available tetrazolium salt based WSTs that include, but not limited to, WST-1, WST-3, WST-4, WST-5, WST-9, WST-10 AND WST-11, MTS and XTT, and an IEA, including mPMS and coenzyme Q1 and the combination of these tetrazolium salts with IEA comprising WST-1+mPMS, WST-3+mPMS, WST-4+mPMS, WST-5+mPMS, WST-9+mPMS, WST-10+mPMS, WST-11+mPMS, XTT+mPMS, MTS+mPMS, WST-3+Q1, WST-4+Q1, WST-5+Q1, WST-9+Q1, WST-10+Q1, WST-11+Q1, XTT+Q1 MTS+Q1.

The term “IKK inhibitor” refers to an agent capable of inhibiting the activity of Inhibitor kappaB kinase (IKK) and thereby inhibiting the kinase activity of IKK and its function of activating NF-κB. Therefore, inhibits NF-κB activity. An IKK inhibitor may be a competitive, noncompetitive, or irreversible IKK inhibitor. “A competitive IKK inhibitor” is a compound or a peptide that reversibly inhibits IKK enzyme activity at the catalytic site; “a noncompetitive IKK Inhibitor” is a compound that reversibly inhibits IKK enzyme activity at a non-catalytic site; and “an irreversible IKK inhibitor” is a compound that irreversibly destroys IKK enzyme activity by forming a covalent bond with the enzyme. The term “IKK inhibitors” include, without limitation, i) compounds previously established to exhibit IKK inhibitory properties including, but not limited to: SPC839 (Signal Pharmaceutical Inc.), Anilino-Pyrimidine Derivative (Signal Pharmaceutical Inc.), PS1145 (Millennium Pharmaceutical Inc.), BMS-345541*(Bristol-Myers Squibb Pharmaceutical Research Institute, IKK inhibitor III), SC-514*(Smithkilne Beecham Corp.), Amino-imidazolecarboxamide derivative (Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives (AstraZeneca), Diarylpybidine derivative (Bayer), Pyridooxazinone derivative (Bayer), Indolecarboxamide derivative (Aventis Pharma), Benzoimidazole carboxamide derivative (Aventis Pharma), Pyrazolo[4,3-c]quinoline derivative (Pharmacia Corporation), Imidazolylquinoline-carbxaldehyde semicarbazide derivative (Tulark Inc.), Pyridyl Cyanoguanidine derivate (Leo Pharma), IkB Kinase Inhibitor Peptide (CalBiochem), IKK-2 Inhibitor IV [5-(p-Fluorophenyl)-2-ureido] thiophene-3-carboxamide (CalBiochem), IKK Inhibitor II, Wedelolactone (CalBiochem), IKK Inhibitor VII (CalBiochem), IKK-2 Inhibitor V N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide IMD-0354 (CalBiochem), IKK-2 Inhibitor VI (5-Phenyl-2-ureido)thiophene-3-carboxamide (CalBiochem), IKK-2 Inhibitor VIII ACHP 2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxyphenyl)-4-(4-piperidinyl)-3-pyridinecarbonitrile (CalBiochem). ii) In a certain embodiment, the group of IKK inhibitors may additionally include compounds discovered to have IKK inhibitory activity, in accordance with the present specification, and previously identified to have anti-tumor activity, including, but not limited to PS1145 (Millennium Pharmaceutical Inc.), BMS-345541*(Bristol-Myers Squibb Pharmaceutical Research Institute).

The term “CK2 inhibitor” represents all protein kinase casein kinase2 inhibitors. The preferred CK2 inhibitors is, but not limited to Apigenin.

The term “Apigenin” CAS Registry Number: 520-36-5, Chemical Abstracts Service Name: 4H-1-benzopyran-4-one,5,7-dihydroxy-2-(4-hydroxy-phenyl)-(9CI). It is also named as Apigenine; Chamomile; Apigenol; Spigenin; and Versulin and is a member of Flavones, a subclass of flavonoids. Apigenin is a multi function signal transductor modulator that reduces DNA oxidative damage; inhibit the growth of human leukemia cells and induced these cells to differentiate; inhibit cancer cell signal transduction and induce apoptosis; act as an anti-inflammatory; and as an anti-spasmodic or spasmolytic. Apigenin inhibits activity of NF-κB, IKK-1 and IKK-2, protein kinase 2 (CK2), mape kinase (MPK), hypoxia inducing factor 1 (HIF), vescular epithelium growth factor (VEGF) and some other molecules and regulatory pathways such as cell cycle and angiogenesis, induce p53 activity, maintaining genomic stability by holding cell cycle for mismatch repair or arrest cell cycle and induce apoptosis etc. Apigenin is know to have the effects of anti-UV radiation caused oxidation, and chemoprevention for cancer. The apigenin, herein, is also described as a representative of the subclasses of flavonoids, the flavones including, but not limited to: tricin, luteolin, tangeritin, 6-hydroxyflavone, Baicalein, Scutellatein, Wogonin, Diosmin, Flavoxate, Chrysin, the glycosided forms of these flavones, and other subclasses of the flavonoids with similar biological activities include, but not limited to Isoflavones, Flavonols, Flavanones, 3-Hydroxyflavanones, Flavan-3-ols, Anthocyanidins, 3-deoxyanthocyanidin, Anthocyanins, Acetylated and glycosides, and Tannins, as well as isoflavonoids and neoflavonoids.

The term “Flavonoids” also called bioflavonoids also collectively know as Vitamin P and citrin, are a class of plant secondary metabolites. Herein flavonoids represent all of the three ketone-containing compounds (flavonoid and flavonols) according to IUPAC nomenclature classifications: i) the flavonoids derived from 2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone) structure; ii) isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure; and iii) neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-1,2-benzopyrone) structure; as well as the non-ketone polyhydroxy polyphenol compounds including: flavanoids, flavan-3-ols and catechins. Sample compounds include, but not limited to Isoflavone:Biochanin A, Daidzein, Daidzin, Formononetin, Genistein, Coumestrol, Puerarin; flavan-3-ols: catechins (catechin, epicatechin (EG), epicatechin, gallate (EGC), and epigallocatechin gallate (EGCG)); flavonol; myricetin, quercetin, and Kaempferol; Isoflavenes: phenoxodiol; Anthocyanins: Antirrhinin, Chrysanthenin, Malvin, Myrtillin, Oenin Primulin, Protocyanin, Tulipanin; 3-deoxyanthocyanidin: Apigeninidin, Columnidin, Diosmetinidin, Luteolinidin, Tricetinidin; Anthocyanidins: Aurantinidin, Cyanidin, Delphinidin, Europinidin, Luteolinidin, Malvidin, Pelargonidin, Peonidin, Petunidin, Rosinidin; 3-Hydroxyflavanones: Dihydrokaempferol, Dihydroquercetin; Flavanones: Eriodictyol, Hesperetin, Homoeriodictyol, Naringenin; Flavonols: Fisetin, Isorhamnetin, Kaempferol, Myricetin, Pachypodol, Quercetin, Rhamnazin, Morin; and their glycoside forms.

The term “HIF” hypoxia inducible factor represents a family of transcription factors that response to decrease of available oxygen or hypoxia in the cellular environment Three family members have been identified. They are HIF-1 (a dimmer composed of HIF-1α and HIF-1β), HIF-2 (a dimmer composed of HIF-2α and HIF-2β), HIF-3 (a dimmer composed of HIF-3α and HIF-3β).

The term “HIF inhibitors” are the biological and non-biological compounds that inhibit HIFs and/or cellular responses to hypoxia, including, but not limited to: 2,2-dimethybenzopyran compounds, chetomin, 2-methoxyestradiol (2ME2), PX-478, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), EZN-2968. camptothecins, NSC 644221, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), rapamycin, and decoy oligonucleotides against HIF-1 RX-0047.

The term “tNOX” represents a tumor specific cell surface NADH oxidase. It is also called ECTO2.

The term “tNOX inhibitors” represents the compounds that are capable of inhibiting the tNOX activity. The tNOX inhibits, herein, includes, but not limited, catechins: catechin, epicatechin (EG), epicatechin gallate (EGC), and epigallocatechin gallate (EGCG); and a isoflavenes analogue derivative, the phenoxodiol.

The term “Oxidative Phosphorylation” is a process that coupling the oxidation of the protons with the synthesis of ATP, which transfer and store the energy derived from glucose metabolism lo the ATP as cellular energy source.

The term “Uncoupler” means to uncouple the cellular oxidative phosphrylation process that blocks the ATP synthesis, the energy metabolism in the cell. The known unucouplers including, but not limited to: dinitrophenol (DNP), Carbonyl cyanide m-chlorophenyl hydra/one (CCCP), Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), Hindered pheniil (SF6847), Salicylanilide S-13, PCP, TTFB, and alpha-(phenylhydrazono)phenylacetonitrile derivatives.

The term “LiCl” is an inorganic salt, lithedium Chloride, and is used as an inhibitor of GSK3β. LiCl, herein, represents the class of inhibitors that inhibit CSK3β.

The term “IKK” represents Inhibitory kappaB Kinase, which phosphorylate IκB that leads to NF-KAPPAB activation. Two IKK isoforms have been identified. They are IKK1 (IKKα) and IKK2 (IKKβ). The term “NF-kappaB” Nuclear factor kappaB is a family of rel proteins that act as transcription factors regulating gene expression. Normally NF-KAPPAB proteins forms a dimmer which also complex with an inhibitory kappa B (IκB) molecule stay in inactive form in the cytoplasm. Upon signal activation, the IκB is phosphorylated by IKK and dissociate from the NF-kappaB dimmer, which release the NF-KAPPAB to entering the nuclear for activating transcription of a special set of genes that are regulated by NF-KAPPAB. The dissociated IκB will be degraded by protesomes. Activation of NF-kappaB favors cell proliferation and survival. NF-kappaB activity has been found to associate with and contribute to carcinogenesis process, tumor progression and resistance of cancer cells to chemo and radiation therapies.

The term “C-Jun N-terminal kinases” (JNKs), originally identified as kinases that bind and phosphosphorylate c-Jun on Ser63 and Ser73 within its transcriptional activation domain, are mitogen-activated protein kinases which are responsive to stress stimuli, such as cytokines, ultraviolet irradiation, heat shock, and osmotic shock, and are involved in T cell differentiation and apoptosis.

The term “Reactive Oxygen Species” (ROS) includes oxygen ions, free radicals and peroxides both inorganic and organic. They are generally very small molecules and are highly reactive due to the presence of unpaired valence shell electrons. ROSs form as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling. The effects of ROS on cell metabolism have been well documented in a variety of species. These include not only roles in programmed cell death and apoptosis, but also positive effects such as the induction of host defence genes and mobilisation of ion transport systems. This is implicating them more frequently with roles in redox signaling or oxidative signaling.

The term “Cancer Cells” represents the cells in culture that were derived from human cancer or tumors, which have malignant features, such as lost of contact inhibition.

The term “Cancer” describes a diseased state in which, a carcinogenic agent or agents causes the transformation of a normal cell into an abnormal cell, the invasion of adjacent tissues by these abnormal cells, and lymphatic or blood-borne spread of malignant cells to regional lymph nodes and to distant sites, i.e., metastasis.

The term “Effective dose” As used herein, the term “effective dose” means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a cell, tissue, system, animal or human that is being sought, for instance, by a researcher or clinician.

The term “therapeutically effective amount” means any amount which, as compared to a corresponding subject who has not received such amount, results in improved treatment, healing, prevention, or amelioration of a disease, disorder, or side effect, or a decrease in the rate of advancement of a disease or disorder. The term also includes within its scope amounts effective to enhance normal physiological function.

The term “Treatment of cancer” describes the drug or reagents administrated lo the cells or to a mammal, the duration of the treatment, the method used to administrate these drugs, or reagents and the order and intervals of between these treatments.

The term “Synergistic effect/Synergize” refers to a combination of two or more treatments, which is more effective to produce advantageous results than the additive effects of these agents.

The term “Chemotherapy Drugs (Agent)” refers to any drugs that have cytrotoxic effect on cancer cells and are currently used as a drug for treating cancer. The drugs that were tested in this specification are listed as the following. Chemotherapy Drags that we are mentioned in this specification were not limit to this list.

The term “5-fluorouracil”, 5-fluoro-2,4-(1H,3H) pyrimidinedione(5-FU), is commercially available as fluorouracil.

The term “Cis-Platinum” cis-diamminedichloroplatinum, is commercially available as PLATINOL™ as an injectable solution.

The term “Paclitaxel” is a potent antineoplastic drug; binds to the N-terminal region of β-tubulin and promotes the formation of highly stable microtubules that resist depolymerization, thus preventing normal cell division and arresting the cell cycle at the G₂/M phase.

The term “Doxorubicin”, (8S,10S)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl, 7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12 naphthacenedione hydrochloride, is commercially available as an injectable form as RUBEX™ or ADRIAMYCIN RDF™.

The term a “therapeutically effective amount” of a compound or a pharmaceutical composition refers to an amount sufficient to modulate cancer cell proliferation in culture, tumor growth or metastasis in an animal, especially a human, including without limitation decreasing tumor growth or size or preventing formation of tumor growth in an animal. This term may also mean the effective amount(s) needed to cause cancer cell death or selective cancer cell death while not causing side effects in normal cells.

The term “Pharmaceutically acceptable” indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

The term a “carrier” refers to, for example, a diluent, adjuvant, excipient, auxiliary agent or vehicle with which an active agent of the present specification is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin. It also include the transfection reagents as used for deliver of DNA and/or RNA into cells either in vitro or in vivo.

The term “Concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

The term “Sequentially” refers to the administration of one active agent used in the method followed by administration of another active agent. After administration of one active agent, the next active agent can be administered substantially immediately after the first, or the next active agent can be administered after an effective time period after the first active agent; the effective time period is the amount of time given for realization of maximum benefit from the administration of the first active agent.

III Targets and Targeting the Therapeutic Targets for the Treatment of Cancer

This description provides nucleotide sequences for genes that implicate and/or can be utilized as therapeutic targets for the treatment of cancer, and polypeptides encoded by such sequences and antibodies and compounds reactive with such polypeptides in methods of treating a cancer, and for agents effective in reducing the activity of cancer-linked genes and thereby treating a cancerous condition which were not previously established for anti-tumor effect(s).

The disclosed nucleotide sequences are related to and derived from a DNA cloning vector, pUC19 (SEQ ID #1), which was discovered to synergize IKK inhibition, inhibit cancer cell growth proliferation and promote cancer cell death when transfection of this vector to cancer cells was combined with or without IKK inhibitor treatment and followed by WST-1r or any of its valid substitutes treatment or in combination with chemotherapeutic drugs. This function of pUC19 has not been previously reported. Other potential DNA sequence may also include a pcDNA3 version 3.1, (SEQ ID 13) and the attached blast result entitled: “NCBI Blast_pcDNA3 Nucleotide sequence (5448 letters)”.

Accordingly, the discovery that the anticancer effect of pUC19 vector (SEQ ID #1) was primarily resides in its DNA sequences that are mapped to transcripts and/or short sequences (from 15 bp up to 100 bp) that flanking the genes in human genome. The human transcripts that pUC19 DNA sequences mapped to are, but not limited to, (1) Homo sapiens transient receptor potential cation channel, subfamily C, member 6 (TRPC6, GeneID; 7225, mRNA: NM_004621.3, SEQ ID #2, #6), (2) Homo sapiens SH3 and PX domains 2B (SH3PXD2B, GeneID: 285590, mRNA:NM_001017995, SeQ ID #3, #7), (3) Homo sapiens membrane associated guanylate kinase, WW and PDZ domain containing 3 (MAGIKK, GeneID: 260425, transcript variant 2, mRNA:NM_152900, SeQ ID #4, #8), (4) the Homo sapiens trans-membrane protein 182 (TMEM 182, GeneID: 130827, mRNA: NM_144632, SeQ ID #5, #9) and (5) Homo sapiens chromosome 6 open reading frame 108 C6orf108, GeneID: 10591 SeQID #14, #15). The human genome sequences that pUC19 DNA sequences mapped to are listed in the attached file “NCBI Blast-pUC19-Human-Transcripts and genome (2686 letters)”, “NCBI Blast_siRNA2 Nucleotide sequence (24 letters)” and “NCBI Blast_pcDNA3 Nucleotide sequence (5448 letters)”.

The polynucleotide disclosed herein incorporate various polynucleotide transcripts (SEQ ID NO: 2, 3, 4, 5 and 14) and, thus, derived amino acid sequence (SEQ ID NO: 6, 7, 8, 9 and 15) from said transcripts are available as targets for treatment of cancer, especially anti-cancer agents, including, with no limitation, peptide and proteins, such as antibodies specific against said polypeptides, peptide inhibitors, small molecule inhibitor, polynucleotides, such as siRNAs, shRNA, anti-sense RNA, anti-sense oligo and dominant negative DNA vectors. In a particular embodiment the wherein said double strand siRNAs are, but not limited to, siRNA1 (SEQ ID #10), siRNA2 (SEQ ID #11), siRNA3 (SEQ ID #12).

The polynucleotides and polypeptides, as gene products, used in the processes may comprise a recombinant polynucleotide or polypeptide, a natural polynucleotide or polypeptide, or a synthetic polynucleotide or polypeptide, or a chemically modified polynucleotide or polypeptide.

The nucleotides and polypeptides of the pUC19 vector, that are mapped to the human genome, flanking genes in the human genome used in the processes of the present description may comprise a recombinant polynucleotide or polypeptide, a natural polynucleotide or polypeptide, or a synthetic polynucleotide or polypeptide.

Fragments of such polynucleotide and polypeptides as are disclosed herein may also be useful in practicing the processes of the present specification. For example, a fragment, derivative or analog of the polynucleotide (SEQ ID#2, 3, 4, 5 and 14) may be substituted by (i) any part of these sequences and/or with mismatches for up to 40% of the total sequences been used for, (ii) fused into a DNA vector or any type of carriers, (iii) nucleotide sequences with modified nucleotides.

Fragments of such polynucleotides and polypeptides as are disclosed herein may also be useful in practicing the processes of the present specification. For example, a fragment, derivative or analog of the polypeptide (SEQ ID NO: 6, 7, 8, 9 and 15) may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (more preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substitute group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as a leader or secretor sequence or a sequence which is employed for purification of the mature polypeptide (such as a histidine hexapeptide) or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

Substituting these siRNAs (SEQ ID 10, 11, 12) as disclosed herein above may also be useful in practicing the processes of the present specification. Examples may include, but not limited to, (i) a siRNA that mapped to another part of the sequence of the coding sequence of the gene, (ii) variations of the siRNA sequences that still capable to target the same gene and reduce it expression level, (iii) any type of modifications of the siRNA either at the nucleotides or the whole siRNA, (iv) put the siRNA sequence into any type of carriers, such as a vector or a chemical for the delivery of the sequence.

The nucleotide sequence of the complete mRNA and open reading frame of the transcripts and amino acid sequences, as discussed above, can be found in the NCBI GenBank database with the Gene ID or accession numbers listed above.

The pharmaceutical compositions and the medical use as described are based, at least in part, on the discovery of inhibitory effect of pUC19 vector in cancer cell growth and proliferation and inducing cancer cell death when combined with IKK inhibitor WST-1r treatment as well as in combination with chemotherapeutic drugs to treat cancer cells. This inhibitory effect of pUC19 DNA transfection may be substituted by siRNA, compounds or small molecule inhibitor, peptide inhibitor, antibody, shRNA, anti-sense RNA, anti-sense oligo, and antibody and dominant negative DNA vectors targeting the gene to alter its expression level, the corresponding transcripts and/or protein as described above in this section and at least in partial by IFN.

Cancers that may be treated using the present discovery include, but are not limited to: cancers of the prostate, colorectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin (including melanoma and basal carcinoma), mesothelial lining, esophagus, breast, lung (including small-cell lung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid, kidney, glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma, choriocarcinoma, cutaneous basocellular carcinoma, and testicular seminoma, sarcoma of muscle, connective tissue or bone and leukemia.

IV. Pharmaceutical Compositions and Methods for Cancer Therapy

I. Inhibition of tPMET and Cell Surface Respiration in Combination with Inhibition of HIF as a Strategy for Synergizing Cancer Cell Death as a Cancer Treatment

A living cell relies on energy. Unlike normal cells that consume oxygen and generate ATP in mitochondrial, cancer cells consume oxygen on cell surface through tPMET. This cellular geographic difference between cancer cells and normal cells makes the PMET a unique site for cancer specific targeting. In addition, cancer cells are resistant to hypoxia due to increased levels and activities of hypoxia inducible factor (HIF). Therefore, blocking the PMET while inhibiting the HIF will induce synergistic and cancer specific cell death for treating cancer in a cancer patient.

One embodiment of the present invention provides pharmaceutical compositions comprising (1) a compound that is impermeable to cell plasma membrane and is capable of interfering, and/or blocking tMPET and/or cell surface respiration, such as WST-1r. WST-3 or their valid substitute, in combination with (2) the second compound that is capable of suppressing cellular survival signaling, such as NF-κB activities, and/or cellular responses to hypoxia, such as apigenin or its valid substitute, HIF inhibitors, IKK inhibitors, flavonoids and pUC19 and its valid substitutes. Such a pharmaceutical composition may be administered, in a therapeutically effective amount, in optimized concentrations in pharmaceutical acceptable medium, to a patient in need for the treatment of cancer.

The first compound that, is composed of two functional chemical groups: A) an functional group that is capable of binding to and/or interfering and/or blocking the electron transport process of the tPMET systems, blocking the coupling of oxidative phosphorylation, and/or inhibiting the tNOX, therefore, to block cell surface respiration and oxygen consumption; and B) another chemical group or a combination of chemical groups that make(s) the entire compound impermeable to cell plasma membrane and capable of blocking the said compound penetrating the cell plasma membrane and entering the cell. By integrating these functional groups into single molecule, the said compound is capable of interfering, inhibiting and/or blocking the tPMET, or the oxidative phosphorylation process or the coupling of the oxidative phosphorylation and cell surface respiration specifically on cell surface, but not affecting the mitochondrial respiration in normal cells.

WST-3 represents such a class of the first compound. It contains a dinitrophenol functional group and a chemical group that is impermeable to cell plasma membrane.

FIG. 1 diagrams the chemical structure of WST-3 (Japanese patent JP,2592436,B, 1995), which is composed with a 2,4-Dinitrophenol (DNP), chemical structure as the said functional chemical group and a [1,3-benzenedilsulfonate]and a [4-lodophenyl] to enhance its hydrophilic feature.

The DNP is an oxidative phosphorylation uncoupler by dissolving in the inner membrane of mitochondria and forms a protonophore, which caused the protons across the mitochondrial membrane, leading to a rapid consumption of energy without generating ATP. By integrating the said DNP with the said second group, the cell plasma impermeable group, it keeps the DNP from entering the cell, but can only act on the cell plasma membrane. As cancer cells respiration mainly rely on cell surface, the WST-3 will only blocks the cell surface respiration of cancer cells, but, will not affect the oxidative phosphorylation in mitochondrial from normal cells, hence, the treatment will be cancer specific.

The DNP as the said first functional chemical group represents an uncoupler of oxidative phosphorylatoin and may also implicate other ways of blocking tPMET and cell surface respiration. Accordingly, the said DNP can be substituted by 1) the compounds of oxidative-phsphorylation decoupling agents comprising: carbonyl cyanide m-chloro phenyl hydrazone (CCCP) and Carbonyl cyanide p-[rifluoromethoxyl]-phenyl-hydrozone (FCCP), SF 6847, salicylanilide S-13, and alpha-(phenylhydrazono)phenylacetonitrile derivatives; and 2) intermediate electron acceptor that direct interact with tPMET, including with no limitation: mPMS and coenzyme Q1; 3) tPMET substrates, such as NADH; 4) the cyanic group (C═N), such as ferricyanide, and respiration inhibitors.

The chemical structure of the said second chemical group or combination of groups Unit keeps the compound impermeable to cell plasma membrane can be designed and/or produced by a skilled person in the field. Examples include, but not limited to the chemical groups that were used for modifying the tetrazolium to form the WSTs, such as the chemical structures of the WST-1, WST-3, WST-4, WST-5, WST-8, WST-9, WST-10, WXST-11, XTT, MSN that keep the compound impermeable to the cell plasma membrane.

Accordingly, the said the first compound is selected from the available groups comprising 1) cell plasma membrane impermeable uncoupler WST-3, 2) tPMET and/or tNOX inhibitors, including, but not limited to capsaicin, capsicin pepper vanilloid, green tea catechin, epigallocatechin-3-gallate; 3) the reagents that interfere tPMET activities including WST-1r and its valid substitutes including but not limited to WST-3+mPM, WST-4+mPMS, WST-5+mPMS, WST-9+mPMS, WST-10+mPMS, WST-11+mPMS, XTT+mPMS, MSN+mPMS, WST-3+Coenzyme Q1, WST-4+Coenzyme Q1, WST-5+Coenzyme Q1, WST-9+Coenzyme Q1, WST-10+Coenzyme Q1, WST-11+Coenzyme Q1, XTT+Coenzyme Q1, and MSN+Coenzyme Q1; 4) the compounds that include at least one of the functional groups as described above in the paragraph [00109] and are impermeable to cell plasma membrane that can be designed and produced by a skilled person in the field.

The said second compound is the one that inhibits cell hepoxia responses, which when combined with the first compound, results in synergistic cell death, such as apigenin. The said second compound is selected from the groups comprising 1) HIF inhibitors, 2) the flavonoids and its subclasses such as flavorones, 3 inhibitors that inhibit NF-κB activities, such including, but no limited to IKK inhibitors, 4) plasmid DNA pUC19 [SEQ ID No:1] and its valid substitutes including, but not limited to at least one of the siRNAs derived from [SEQ ID No: 10, 11, 12], means to targeting the genes [SEQ ID No: 2, 3, 4, 5, 14] and their corresponding gene products [SEQ ID No: 6, 7, 8, 9, 15] to alter their expression levels and functional activities including, but not limited to nucleotide sequences including dominant negative DNA that block the function of the corresponding gene products, siRNA, antisense RNA, antisense oligo, peptides, peptide inhibitors, antibodies, small molecule inhibitors.

The said apigenin is a flavone, a subclass of flavonoids, and is a multi-function signal transduction modulator and/or inhibitor to cells. Its function includes, but not limited to induction of p53 activation, suspend cell cycle progression for maintaining genomic stability, inhibiting expression and/or activities of hypoxia induced factor-1 (HIF-1), casein kinase II, NF-□B, IKK and induction of generation of reactive oxygen species (ROS) and more.

The second compound and the valid substitutes of apigenin is selected from the groups comprising (I) At least one flavones, include, but not limited to nature existed flavones, such as: tricin, Luteolin, Tangeritin, Chrysin, 6-hydroxyflavone, Baicalein, Scutellarein, Wogonin and synthetic flavones, such as: Diosmin, Flavoxate, additional subgroups of flavones:flavonols, flavannones, flacanonols, catechins, isoflavones; or

(2) at least one from other subgroups of Flavonoid (Bioflavonoids) and their isoforms including naturally existed, artificial modified ketone isoforms and synthetic compounds including, but not limited to flavonoids, derived from 2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone) structure (examples: quercetin, rutin); isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure: neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-1,2-benzopyrone) structure, and flavonoids as a non-ketonepolyhydroxy polyphenol compounds, including: flavanoids, flavan-3-ols and catechins. Sample compounds include, but not limited to Isoflavone-Biochanin A, Daidzein, Daidzin, Formononetin, Genistein, Coumestrol, Puerarin; flavan-3-ols: catechins (catechin, epicatechin (EG), epicatechin, gallate (EGC), and epigallocatechin gallate (EGCG)); flavonol: myricetin, quercetin, and Kaempferol; Isoflavenes: phenoxodiol; Anthocyanins: Antirrhinin, Chrysanthenin, Malvin, Myrtillin, Oenin Primulin, Protocyanin, Tulipanin; 3-deoxyanthocyanidin: Apigeninidin, Columnidin, Diosmetinidin, Luteolinidin, Tricetinidin; Anthocyanidins: Aurantinidin, Cyanidin, Delphinidin, Europinidin, Luteolinidin, Malvidin, Pelargonidin, Peonidin, Petunidin, Rosinidin: 3-Hydroxyflavanones: Dihydrokaempferol, Dihydroquercetin; Flavanones: Eriodictyol, Hesperetin, Homoeriodictyol, Naringenin; Flavonols: Fisetin, Isorhamnetin, Kaempferol, Myricetin, Pachypodol, Quercetin, Rhamnazin, Morin; and their glycoside forms; or

(3) At least one HIF inhibitors and/or inhibition of cellular responses to hypoxia including, but not limited to: 2,2-dimethybenzopyran compounds, chetomin, 2-methoxyestradiol (2ME2), PX-478, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), EZN-2968, camptothecins, NSC 644221, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), rapamycin, and decoy oligonucleotides against HIF-1 RX-0047; or

(4) IKK inhibitors are as listed above and following embodiments include compounds which exhibits IKK inhibitory activity in pharmaceutically acceptable medium. The at least one IKK inhibitor may be selected from compounds of the group consisting of, without limitation, i) compounds previously established to exhibit IKK inhibitory properties including, but not limited to: SPC839 (Signal Pharmaceutical Inc.), Anilino-Pyrimidine Derivative (Signal Pharmaceutical Inc.), PS1145 (Millennium Pharmaceutical Inc.), BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute), SC-514*(Smithkilne Beecham Corp.), Amino-imidazolecarboxamide derivative (Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives (AstraZeneca), Diarylpybidine derivative (Bayer), Pyridooxazinone derivative (Bayer), Indolecarboxamide derivative (Aventis Pharma), Benzoimidazole carboxamide derivative (Aventis Pharma), Pyrazolo[4,3-c] quinoline derivative (Pharmacia Corporation), Imidazolylquinoline-carbxaldehyde semicarbazide derivative (Tulark Inc.), Pyridyl Cyanoguanidine derivate (Leo Pharma), IκB Kinase Inhibitor Peptide (CalBiochem), IKK-2 Inhibitor IV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (CalBiochem), IKK Inhibitor II (Wedelolactone (CalBiochem), IKK Inhibitor VII (CalBiochem), IKK-2 Inhibitor V (N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide IMD-0354, CalBiochem), IKK-2 Inhibitor VI (5-Phenyl-2-ureido)thiophene-3-carboxamide, CalBiochem), IKK-2 Inhibitor VIII (ACHP 2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxyphenyl)-4-(4-piperidinyl)-3-pyridinecarbonitrile, CalBiochem). ii) In a certain embodiment, the group of IKK inhibitors may additionally include compounds discovered to have IKK inhibitory activity, in accordance, and previously identified as anti-tumor agents, including, but not limited to PS1145 (Millennium Pharmaceutical Inc.), BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute); or

(5) at least one nucleotide sequences [SEQ ID NO: 1, 10, 11, 12, 13], and means for targeting the genes of polynucleotide sequences [SEQ ID No: 2, 3, 4, 5, 14] and peptide sequences [SEQ ID No: 6, 7, 8, 9, 15] to inhibit the expression levels and functional activities of the corresponding genes by siRNA, antisense RNA, antisense oligo, dominant negative DNA, peptide, peptide inhibitors, antibodies, small molecule inhibitors.

2. Pharmaceutical Composition and Method of WST-3 And Apigenin Combination Treatment for Cancer Therapy

One of the best mode embodiment of the present invention provides pharmaceutical compositions comprising (1) at least one Water-soluble tetrazolium salts 3 (WST-3,2-(4-Iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, sodium salt, FIG. 1A) or its valid substitutes in combination with (2) at least one apigenin or its valid substitutes. Such a pharmaceutical composition may be administered, in a therapeutically effective amount, in optimized concentration in pharmaceutical acceptable medium, to a patient in need for the treatment of cancer.

WST-3 is a water soluble tetrazoliums (WST) that were developed by Dojindo Inc. whose WSTs have sulfate groups added directly or indirectly to the phenyl ring to improve water-solubility that also makes the compound impermeable to cell plasma membrane. Different from all other WSTs, WST-3 contains a 2,4-dinitrophenol (DNP) group directly linked to the tetrazolium ring (FIG. 1).

DNP, a cellular metabolic poison, represents a class of six manufactured chemical compounds that can dissolve in the mitochondria membrane, acts as a proton ionophore, an agent that can shuttle protons (hydrogen ions) across biological membranes, where it uncouples oxidative phosphorylation by carrying protons across the mitochondrial membrane, leading to a rapid consumption of energy without generating ATP. DNP defeats the proton gradient across mitochondria and chloroplast membranes, collapsing the proton motive force that the cell uses to produce most, of its ATP chemical energy. Instead of producing ATP, the energy of the proton gradient is lost as heat. Cells counteract the lowered yields of ATP by oxidizing more stored reserves such as carbohydrates and fat DNP has been used as weight loss treatment for burning extra fats. However, it is toxic to the cells by exoughsting cell energy sources.

General structure feature of uncouplers are weak acids comprising the chemical groups: Weakly Acidic Phenols, benzimidazoles, N-phenylanthranilates, salicylanilides, phenylhydrazones, salicylic acids, acyldi-thiocarbazates, cumarines, and aromatic amines.

The chemical structures of representative weakly acidic uncouplers that are capable of substituting the DNP are selected from the groups comprising: S-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanilide (S-13), sodium 2,3,4,5,6-pentachlorophenolate (PCP), 4,5,6,7-tetrachloro-2-(trifluoromethyl)-1H-benzimidazole (TTFB), Flufenamic acid (2-[3-(trifluoromethyl)anilino]benzoic acid), 3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF6847), carbonyl cyanide m-chloro phenyl hydrazone (CCCP) and Carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP), and alpha-(phenylhydrazono)phenylacetonitrile derivatives.

The incorporating DNP into the water soluble tetrozolium salts that keeps the WST-3 impermeable to cell plasma membrane, hence, makes WST3 capable of mimicking the DNP effect to act on cell plasma membrane for uncoupling oxidative phosphorylation that interrupts tPMET, but does not affect mitochondria in normal cells (FIG. 1).

Thus, WST-3 represents classes of compounds that comprises of (1) an active group that is capable of blocking tPMET and/or oxidative phosphorylation and/or the coupling process between these two processes and (2) the chemical structure that keeps the compound impermeable to cell plasma membrane. In this way such a compound shall be able to specifically block the tPMET electron transfer and and/or oxidative phosphorylation of ADP on cell surface, hence, specifically inhibit tPMET and ATP production in cancer cells.

The valid substitutes of WST-3 include, but not limited to the compounds that contains the combination of the two said features including (1) the active group as described above that can block the tPMET and/or oxidative phosphorylation and/or the coupling of the tPMET and the oxidative phosphoryalation process (2) the chemical structure that makes the resulting compound impermeable to cell plasma membrane as described above for the first compound as described in paragraph (0038).

The said Apigenin is a flavonoid and is a multi-function inhibitor to cells. Its function includes, but not limited to induction of p53 activation, suspend cell cycle progression to maintain genomic stability, inhibiting expression and/or activities of hypoxia induced factor-1 (HIF-1), casein kinase II, NF-□B, induction of generation of ROS and more.

The valid substitutes of apigeninare are selected from the groups comprising: at least one flavones, include, but not limited to nature existed flavones, such as: Tricin, Luteolin, Tangeritin, Chrysin, 6-hydroxyflavone, Baicalein, Scutellarein, Wogonin and synthetic flavones, such as: Diosmin, Flavoxate, additional subgroups of flavones: flavonols, flavannones, flacanonols, catechins, isoflavones; at least one from other subgroups of Flavonoid or Bioflavonoids and their isoforms including naturally existed, artificial modified isoforms and synthetic compounds including, but not limited to□ flavonoids, derived from 2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone) structure (examples: quercetin, rutin); isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure; neoflavonoids derived from 4-phenylcoumarine (4-phenyl-1,2-benzopyrone) structure, and flavanoids as a non-ketonepolyhydroxy polyphenol compounds as described in paragraph [00115 and 00114]; at least one HIF inhibitors and/or inhibition of cellular responses to hypoxia including, but not limited to: 2,2-dimethybenzopyran compounds, chetomin, 2-methoxyestradiol (2ME2), PX-478, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), EZN-2968, camptothecins, NSC 644221, 3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), rapamycin. and decoy oligonucleotides against HIF-1 RX-0047.

IKK inhibitors are as listed above and following embodiments include compounds which exhibits IKK inhibitory activity in pharmaceutically acceptable medium. The at least one IKK inhibitor may be selected from compounds of the group consisting of, without limitation, i) compounds previously established to exhibit IKK inhibitory properties including, but not limited to: SPC839 (Signal Pharmaceutical Inc.), Anilino-Pyrimidine Derivative (Signal Pharmaceutical Inc.), PS1145 (Millennium Pharmaceutical Inc.), BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute), SC-514*(Smithkilne Beecham Corp.), Amino-imidazolecarboxamide derivative (Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives (AstraZeneca), Diarylpybidine derivative (Bayer), Pyridooxazinone derivative (Bayer), Indolecarboxamide derivative (Aventis Pharma), Benzoimidazole carboxamide derivative (Aventis Pharma), Pyrazolo[4,3-c] quinoline derivative (Pharmacia Corporation), Imidazolylquiuoline-carbxaldehyde semicarbazide derivative (Tulark Inc.), Pyridyl Cyanoguanidine derivate (Leo Pharma), IκB Kinase Inhibitor Peptide (CalBiochem). IKK-2 inhibitor IV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (CalBiochem), IKK Inhibitor II (Wedelolactone (CalBiochem), IKK Inhibitor VII (CalBiochem), IKK-2 Inhibitor V (N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide IMD-0354, CalBiochem), IKK-2 Inhibitor VI (5-Phenyl-2-ureido)thiophene-3-carboxamide, CalBiochem), IKK-2 Inhibitor VIII (ACHP 2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxyphenyl)-4-(4-piperidinyl)-3-pyridinecarbonitrile, CalBiochem), ii) In a certain embodiment, the group of IKK inhibitors may additionally include compounds discovered to have IKK inhibitory activity, in accordance, and previously identified as anti-tumor agents, including, but not limited to PS1145 (Millennium Pharmaceutical Inc.), BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute).

(5) at least one nucleotide sequences [SEQ ID NO:1, 10, 11, 12, 13], and means for targeting the genes of polynucleotide sequences [SEQ ID No: 2, 3, 4, 5, 14] and peptide sequences [SEQ ID No: 6, 7, 8, 9, 15] to inhibit the expression levels and functional activities of the corresponding genes by siRNA, antisense RNA, antisense oligo, dominant negative DNA, peptide, peptide inhibitors, antibodies, small molecule inhibitors.

One embodiment provides methods and a treatment protocol for inducing cancer cell death and tumor suppression to treat cancer in a patient. In accordance with this method, it has been discovered that the combination of WST-3 and/or its valid substitutes with an apigenin and/or its valid substitutes for synergistic induction of cancer cell death and suppression of tumor growth.

Accordingly, cancer cells are treated with effective dose(s) of WST-3 and/or at least one of its valid substitutes in combination with apigenin and/or al least one of its valid substitutes in pharmaceutical acceptable medium for effective time period.

The valid substitutes of WST-3 include, but not limited to the compounds that contains the combination of the two said features including (1) the active group as described above that can block the tPMET and/or oxidative phosphorylation and/or the coupling of the tPMET and the oxidative phosphorylation process (2) the chemical structure that makes the resulting compound impermeable to cell plasma membrane as described above for the first compound as listed in the paragraph (0038).

The suitable active groups that can block the tPMET and oxidative phosphorylation include, but not limited to the DNP group and the cyano group.

Suitable as least one valid substitute for apigenin, as listed above, include, but not limited to (1) any other flavonoids and their isoforms including naturally existed, artificial modified isoforms and synthetic compounds, any isoflevens as described in paragraph [00114 and 00115]; (3) inhibitors to HIF-1 and/or any inhibitors to cellular responses to hypoxi as described in paragraph [00116]; (4) inhibitors to NOXes especially tNOX as described in paragraph [0035]; (5) inhibitors that can mimic one or more of the predetermined apigenin effects.

It is yet another embodiment to treat cancer cells with WST-3 with at least one apigenin or any of its valid substitutes for both WST-3 and apigenin simultaneously and sequentially in any order for each of the above and following embodiments, forming a more preferred embodiment.

It is yet another embodiment to treat cancer cells with WST-3 with at least one IKK inhibitor or all other valid substitutes for both WST-1r and IKK inhibitor simultaneously and sequentially in any order for each of the above and following embodiments, forming a more preferred embodiment.

The in vitro effective dose of WST-3 may be 50 μM or lower, but can be higher as well.

The effective dose of apigenin under in vitro cell culture may be at 1-100 μM.

The WST-3 or at least one of its valid substitutes and apigenin or at least one of its valid substitutes may be administered to cancer cells or to cancer patients concurrently, separately and/or sequentially in any order.

Each of the treatment agents may be administrated via oral intra peritonea injection, intra muscular injection, intra venous injection, intra venous infusion, intra artery infusion, intra artery injection, as well as via dermal penetration.

The treatment time of WST-3 or at least one of its valid substitutes may be between pulsed for 30 minutes to 8 hours of initial treatment or continuesly.

The treatment time of apigenin or at least one of its valid substitutes may be last for 15 min to 24 hours consecutively or longer.

In other words, the WST-3 or at least one of its valid substitutes may be treated first with effective dose for 30 minutes to 4 hours in the absence of apigenin, then, remove the WST-3 and administer the apigenin or at least one of its valid substitutes to the cancer cells for another 4 to 24 hours; or

Alternatively, administering the apigenin or at least one of its valid substitutes to the cancer cells for another 4 to 24 hours, and then, remove the administer the apigenin or at least one of its valid substitutes and administering the WST-3 or its valid substitutes to cancer cells for 30 minutes to 4 hours; or

Alternatively, administering the apigenin or at least one of its valid substitutes and the WST-3 or its valid substitutes to the cancer cells for 30 minutes to 4 hours, then remove the treatments and administering the apigenin or at least one of its valid substitutes for another 4-24 hours, or

Alternatively, administering the apigenin or at least one of its valid substitutes for 24 hours, then, administering the WST-3 or at least one of its valid substitutes to the treatment of cancer cells for 30 minutes to 4 hours,

Alternatively, administering of apigenin or at least one of its valid substitutes and the WST-3 or its valid substitutes can be concurrently to the cancer cells for 30 minutes to 4 hours.

Alternatively, administering of apigenin or at least one of its valid substitutes and the WST-3 or its valid substitutes can be concurrently to the cancer cells continuesly.

The actual treatment doses of WST-3 and apigenin and the treatment time of these compounds can be adjusted by a physician or a skilled person.

The preferred embodiment for the treatment is lo administer the apigenin or at least one of its valid substitutes and the WST-3 or its valid substitutes to the cancer cells for 4 hours, then remove the treatments and administering the apigenin or at least one of its valid substitutes for another 24 hours. This is because we have the most date for.

Cancers that may be treated using the combinatorial protocol with WST-3 or its valid substitutes in combination with apigenin or its valid substitutes are carcinomas and sarcomas include, but are not limited to those carcinomas and sarcomas that may be treated using the present protocol include, but are not limited to: cancers of the sqoumas cell carcinoma, breast, prostate, colorectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin (including melanoma and basal carcinoma), mesothelial lining, esophagus, breast, muscle, connective tissue, lung (including small-cell lung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid, kidney, or bone; glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma, sarcoma, choriocarcinoma, cutaneous basocellular carcinoma, and testicular seminoma, soft tissue sarcoma, as well as lymphomas and leukemia.

Accordingly, one of the embodiments of this invention provides a method for treating cancer in a patient by combination of (1) means of blocking tPMET and/or uncoupling the oxidative-phosporylation on the cell plasma membrane with (2) means of inhibiting cellular responses to hypoxia, HIF, NOX, NF-κB activity or mimic one or more of predetermined apigenin effects on cancer cells.

The means to block tPMET and/or uncouple oxidative phosporylation on the cell plasma membrane include, but not limited to: the cell plasma membrane impermeable tMPET oxidative phosphorylation uncoupler or its valid substitutes are the compounds that can inhibit the trans plasma membrane electron transfer process, or the oxidative phosphorylation process or the coupling of electron transport and the oxidative phosphorylation and impermeable to cell plasma membrane.

The means of inhibiting cellular responses to hypoxia, HIF, NOX, NF-κB activity, or mimic one or more of predetermined apigenin effects on cancer cells including, but not limited to treatment with apigenin, or its valid substitutes.

The order of the treatment to cancer cells or cancer patients of the means of blocking tPMET and/or oxidative phosporylation on the cell plasma membrane with the means of inhibiting cellular responses to hypoxia HIF, NOX, NF-κB activity, or mimic one or more of predetermined apigenin effects on cancer cells can be concurrently or sequentially in any order at effective doses and effective time period for the treatment.

The present invention also provides additional methods for inducing cancer cell death and suppressing tumor growth in cancer patients. In accordance with the present invention, it has been discovered that the combination of a flavonoid, apigenin, or its valid substitutes, with the WST-3 or the valid substitutes at effective concentration for synergistic induction of cancer cell death. Accordingly, the present invention provides a pharmaceutical composition and protocol for the treatment of cancer in a patient in need with effective dose comprising of at least one flavonoid, specifically, apigenin, or its valid substitutes, with WST-3 or at least one of the valid substitutes of the WST-3 in a pharmaceutical acceptable medium.

Suitable flavonoids include, but not limited to, apigenin and valid substitutes of apigenin as described above in paragraph [00114-00118] in pharmaceutically acceptable medium.

The valid substitutes of apigenin include the compounds that exhibit inhibitory activity as at least one of the effects of that Apigenin does in pharmaceutically acceptable medium.

The suitable at least one of the valid substitutes for the WST-3, as noted herein above in paragraph [00111], include, but are not limited to the individual components that are comprises the active group as represented by DNP and the valid substitutes for tetrazolium salts that make the compound impermeable to cell plasma membrane at optimized concentrations in pharmaceutically acceptable medium.

The effective concentration of apigenin that were used may vary depending on cell type. The preferred dose is at the range of 1-100 μM in vitro.

For all the above and following embodiments, the effective concentration of WST-3 and the valid substitutes may vary depending on the individual composition and the effective concentration of each of the composition may or may not be the same concentration as that in the WST-3 and may vary from each of the compositions and their valid substitutes and between in vitro and in vivo usage. The preferred in vitro concentration range tor in vitro treatment of WST-3 is 50 μM or lower in a pharmaceutical acceptable medium.

In a specific embodiment of the present invention, the administration of the WST-3 or at least one valid substitutes of WST-3, the apigenin or at least one of the valid substitutes of apigenin can be in any type of order. Specifically, the WST-3 or at least one valid substitutes of WST-3, and the apigenin or at least one of the valid substitutes, of apigenin may be administered to the cells or patient concurrently or sequentially. In other words, the apigenin or at least one of the valid substitutes of apigenin or the WST-3 or the at least one substitute of WST-3 may be administered first, or the WST-3 or at least one valid substitutes of WST-3, and the apigenin or at least one of the valid substitutes of apigenin may be administered at the same time. The preferred order of the treatment in this invention is to administer the WST-3 or the valid substitutes of WST-3 and the apigenin or the valid substitutes of apigenin simultaneously and then, after removal of the WST-3, add apigenin again and keep in contact with cells for another 24 hours.

In a particular embodiment, the treatment of WST-3 is in contact with cells for 15 minutes to 8 hours. The preferred time is between 30 min to 4 hours. The more preferred time is between 2-4 hours. A removal of the WST-3 or its valid substitute's from treatment is required for all the above and following embodiments to induce programmed cell death of the treated cells by this method thereof.

Moreover, the present invention provides a method for the treatment of cancer by administering to a patient, in need thereof, a therapeutically effective dose of at least one of the WST-3 or its valid substitutes and apigenin or at least one of its valid substitutes mentioned above in pharmaceutical acceptable medium.

Cancers that may be treated using the combinatorial protocol with WST-3 or its valid substitutes in combination with apigenin include, but are not limited to Cancers that may be treated using the present protocol include, but are not limited to: colorectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin (including melanoma and basal carcinoma), mesothelial lining, white esophagus, breast, muscle, connective tissue, lung (including small-cell lung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid, kidney, or bone; glioblastoma, mesothelioma renal cell carcinoma, gastric carcinoma sarcoma, choriocarcinoma, cutaneous basocellular carcinoma, and testicular seminoma, leukemia lymphoma and sarcomas, lymphomas and leukemia.

3. Pharmaceutical Composition and Treatment Method of Combination of WST-1r and Apigenin for the Treatment Of Cancer

One embodiment of the invention provides pharmaceutical compositions comprising 1) WST-1r or its valid substitutes, which have not previously been established as having an anticancer effect. The WST-1r has been used as a cell proliferation detection agent, the Cell Proliferation-WST-1. When WST-1r combined with 2) apigenin, a flavonoid, or its valid substitutes, or an IKK inhibitor, or trasnfection of Puc19 or its valid substitutes synergize the induction of cancer cell death. Such a pharmaceutical composition may be administered, in a therapeutically effective amount, in optimized concentration in phosphate buffered saline or any of the valid pharmaceutical acceptable medium, to a patient hi need for the treatment of cancer.

The afficacy of the said anticancer treatment immediate above was synergized by combination use of WST-1r and its valid substitutes which have not previously been established. The Cell Proliferation WST-1r is composed of a tetrazolium salt, WST-1c (WST-1, Ishiyam M, et al Biol Pharm Bull 1996, 19:1515-20; Berridge M V, et al Biotechnology Annual Review, Vol. 11:127-152, 2005), and an IEA, mPMS, (Berridge M V, et al Biotechnology Annual Review, Vol. 11:127-152, 2005) diluted in phosphor buffered saline. WST-1r has also been used for measuring tPMET activity. Treatment with WST-1r enhanced cell respiration. When the WST-1r treatment was withdraw following the treatment and in combination of inhibiting HIF by apigenin or any of its valid substitutes resulted in synergized cancer cell death. In the present invention, WST-1r is used as a drug for a combination treatment for cancer therapy.

In accordance, the active gradient of WST-1r for the treatment of cancer can be either the WST-1c or the mPMS or the combination of the two components in optimized concentration and optimized ratio. The WST-1r that as described herein above and there after represents a group of chemical compound or mixture of combinations of a water soluble tetrazolium salt and an IEA that are capable of interacting with and/or interfering to tPMET, and/or capable of inducing reactive oxygen species (ROS) generation.

The valid substitutes of WST-1c include, but not limited to other WST, including, but not limited to WST-3, WST-4, WST-5, WST-9, WST-10, WST-11, MSN and XTT at optimized concentration in a pharmaceutical acceptable medium.

The valid substitutes of mPMS include, other IEAs, examples may be as, but not limited to coenzyme Q1 (Berridge M V, et al Biotechnology Annual Review, Vol. 11:127-152, 2005) at optimized concentration in a pharmaceutical acceptable medium.

The WST-1r includes compositions of at least one WST, WST-1c, and at lease one IEA, mPMS in optimized concentration and ratio in a pharmaceutical acceptable medium.

The valid substitute of WST-1r includes, but not limited to (1) the combination of at least one WST with at least one IEA. Examples as, but not limited to: WST-1m+PMS, WST-3+mPMS, WST-4+mPMS WST-5+mPMS, WST-9+mPMS, WST-10+mPMS, WST-11+mPMS, MSN+mPMS XTT+mMS, WST-1+coenzyme Q1, WST-3+coenzyme Q1, WST-4-+coenzyme Q1 WST-5+coenzyme Q1, WST-9+coenzyme Q1, WST-10+coenzyme Q1, WST-11+coenzyme Q1, MSN+coenzyme Q1 XTT+coenzyme Q1; (2) at least one of the WST, such as, with no limitation, WST-3; (3) at least one IEA, such as, with no limitation, mPMS and coenzyme Q1 at optimized concentration in a pharmaceutically acceptable medium.

The Apigenin herein represents the second molecule of this combination composition. The valid substitutes of apigenin are selected from the groups comprising: 1) at least one flavone as listed above in paragraph [00114], 2) at least one flavonoids or isoflavonoids as listed above in paragraph [00115]; 3) at least one HIF inhibitors as described above in paragraph [00116], 3) at least one IKK inhibitors as described above in paragraph [00117], 4) at least one nucleotide sequences [SEQ ID NO:1, 10, 11, 12, 13], and means for targeting the genes of polynucleotide sequences [SEQ ID No: 2, 3, 4, 5, 14] and peptide sequences [SEQ ID No: 6, 7, 8, 9, 15] as listed above in paragraph [00118].

It is yet another embodiment to treat cancer cells with WST-1r and apigenin, a flavonoids or all other valid substitutes for both WST-1r and apigenin simultaneously and sequentially in any order for each of the above and following embodiments, forming a more preferred embodiment.

It is yet another embodiment to treat cancer cells with WST-1r with at least one IKK inhibitor or all other valid substitutes for both WST-1r and IKK inhibitor simultaneously and sequentially in any order for each of the above and following embodiments, forming a more preferred embodiment.

It is yet another embodiment to treat cancer cells with (1) the DNA transfection, or IFN, or siRNA transfection or all other valid substitutes and, then, (2) one of the IKK, or CK2 or GSK3β inhibitors and, treat with WST-1r simultaneously or sequentially in any order for each of the above and following embodiments, forming a more preferred embodiment.

It is yet another embodiment to treat with (1) the DNA transfection, or IFN, or siRNA transfection or all other valid substitutes and, then, (2) one of the IKK, or CK2 or GSK3β inhibitors and, treat with electron coupling reagent of the WST-1r simultaneously or sequentially in any order for each of the above and following embodiments, forming a more preferred embodiment

It is yet another embodiment to treat with (1) the DNA transfection, or IFN, or siRNA transfection or all other valid substitutes and, then, (2) one of the IKK, or CK2 or GSK3β inhibitors and, simultaneously or sequentially in any order treat with all the remaining subcomponent of the WST-1r for each of the above and following embodiments, forming a more preferred embodiment

It is yet another embodiment to treat with (1) the DNA transfection, or IFN, or siRNA transfection or all other valid substitutes and, then, (2) one of the IKK, or CK2 or GSK3β inhibitors and, simultaneously or sequentially in any order treat with any valid substitution for WST-1r for each of the above and following embodiments, forming a more preferred embodiment

It is yet another embodiment to treat with (1) the DNA transfection, or IFN, or siRNA transfection or all other valid substitutes and, then, (2) one of the IKK, or CK2 or GSK3β inhibitors and, simultaneously or sequentially in any order treat with any valid substitution for WST-1c for each of the above and following embodiments, forming a more preferred embodiment

It is yet another embodiment to treat with (1) the DNA transfection, or IFN, or siRNA transfection or all other valid substitutes and, then, (2) one of the IKK, or CK2 or GSK3β inhibitors and, simultaneously or sequentially in any order treat with any valid substitution tor electron coupling reagent of the WST-1r, such as mPMS, for each of the above and following embodiments, forming a more preferred embodiment.

It is yet another embodiment to treat with (1) the DNA transfection, or IFN, or siRNA transfection or all other valid substitutes and, then, (2) one of the IKK, or CK2 or GSK3β inhibitors and, simultaneously or sequentially in any order treat with any valid substitution tor the remaining subcomponent of the WST-1r for each of the above and following embodiments, forming a more preferred embodiment.

It is yet another embodiment to treat with (1) the DNA transfection, or IFN, or siRNA transfection or all other valid substitutes and, then, (2) one of the IKK, or CK2 or GSK3β inhibitors and, treat with WST-1r simultaneously or sequentially in any order treat with any valid substitution as any type of combination of the valid substitutes and the subcomponent of the WST-1r for each of the above and following embodiments, forming a more preferred embodiment.

Moreover, the present descriptions provide pharmaceutical compositions and methods for the treatment of cancer by administering to a patient, in need thereof, a therapeutically effective amount of at least one of the WST-1r component or its valid substitutes mentioned immediately above.

The optimized concentration may or may not be the same concentration as that of the Cell Proliferation WST-1 reagent and may vary from each of the compositions and their valid substitutes and between in vitro and in vivo usage. The preferred optimized in vitro WST-1r, WST-3+mPMS, WST-4+mPMS and WST-3 are the most preferred embodiment because they were the component for which we have the most valid data.

Moreover, the present description provides a method for the treatment of cancer by administering to a patient, in need thereof, a therapeutically effective amount of at least one of the WST-1r component or its valid substitutes mentioned immediately above.

In a particular embodiment, the preferred treatment of WST-1r is in contact with cells for at lease 15 minutes or longer. The more preferred treatment time tor WST-1r is between 30 min to 4 hours. The even more preferred treatment time for WST-1r is between 2-4 hours.

Each of the treatment agents may be administrated via oral, intra peritonea injection, intra muscular injection, intra venous injection, intra venous infusion, intra artery infusion, intra artery injection, as well as via dermal penetration.

Cancers that may be treated using the present protocol include, but are not limited to: carcinoma derived from prostate, colorectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin (including melanoma and basal carcinoma), mesothelial lining, white esophagus, breast, muscle, connective tissue, lung (including small-cell lung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid, kidney, or bone; glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma, sarcoma, choriocarcinoma, cutaneous basocellular carcinoma, and testicular seminoma, leukemia, lymphoma and sarcomas.

4. Combinatorial Therapies with Inhibitors and WST-1r for the Treatment of Cancer

The present description provides additional methods for inducing cancer cell death for the treatment of cancer for a patient in need. In accordance, it has been discovered that the combination of pUC19 DNA transfection and/or its valid substitutes with an IKK inhibitor plus WST-1r or its valid substitutes for synergistic inducing cancer cell death. Accordingly, the present description provides a pharmaceutical composition and protocol for the treatment of cancer in a patient comprising at lease pUC19 DNA transfection or its valid substitutes in combination with at least one IKK inhibitor and WST-1r or at least one of the valid substitutes of the WST-1r. Also provided is a method for treating cancer in a patient by IFN in combination with administering an effective amount of at least one IKK inhibitor and WST-1r or at least one of the valid substitutes of the WST-1r. Also provided is a method for treating cancer in a patient by transfection of the cells with siRNA in combination with administering an effective amount of at least one IKK inhibitor and WST-1r or at least one of the valid substitutes of the WST-1r.

The DNA transfection may be substituted by (i) administering a suitable dose of at least one IFN, or (ii) transfection of at least one specific siRNA targeting at least one of the target transcripts as described previously in this description, or (iii) chemical compounds or small molecule inhibitors that targets at least one of the target genes and/or its gene products as described previously in this description, or (iv) antibodies targeting at least one of the target genes products as described previously in this description, (v) anti-sense RNAs targeting at least one of the target transcripts as described previously in this description, (vi) shRNAs targeting at least one of the target transcripts as described previously in this description, (vii) anti-sense oligos targeting at least one of the target transcripts as described previously in this description, (viii) A dominant negative DNA vector targeting at least one of the target genes as described previously in this description, (ix) peptides targeting at least one of the target genes products as described previously in this description.

The target genes are, but not limited to, (1) Homo sapiens transient receptor potential cation channel, subfamily C, member 6 (TRPC6, GeneID: 7225,), mRNA (NM_004621.3) synonyms: TRP6, FSGS2, FLJ11098 (SEQ ID #2, #6), (2) Homo sapiens SH3 and PX domains 2B (SH3PXD2B), mRNA (.(SH3PXD2B, GeneID: 285590), mRNA (NM_001017995) synonyms: HOFI; FLJ20831; KIAA1295 (SEQ ID #3, #7), (3) Homo sapiens membrane associated guanylate kinase, WW and PDZ domain containing 3 (MAGIKK, GeneID: 260425), transcript variant 2, mRNA (NM_152900.1) synonyms: MAGI-3, MGC163281 (SEQ ID #4, #8), and (4) the Homo sapiens transmembrane protein 182 (TMEM 182, GeneID: 130827), mRNA (NM_144632.2) (SEQ ID #5, #9).

The gene products include, but not limited to, the transcripts from these genes and proteins above.

The siRNA sequences and the targets of the siRNA sequences may also include the human genomic sequences that flanking the genes as listed in the attached file entitled: “NCBI Blast-pUC19-Human-Transcripts and genome (2686 letters)”, “NCBI Blast_siRNA2 Nucleotide sequence (24 letters)” and “NCBI Blast_pcDNA3 Nucleotide sequence (5448 letters)”. NCBI Blast-pUC19-Human-Transcripts and genome.

The at least one IFN may be selected from the subfamily of type I IFN including, but not limited to: IFNα A, IFNα B, IFNα C, IFNα D, IFNα F, IFNα G, IFNα H, IFNα I, IFNα J, IFNα K, IFNα 4b, IFNα WA, and IFNα.

The effective concentration of IFN that were used for treating cancer cells was 10 unit/ml or lower for each IFN used.

Suitable IKK inhibitors include any compound which exhibits IKK inhibitory-activity.

The at least one IKK inhibitor may be selected from compounds of the group consisting of, without limitation, i) compounds previously established to exhibit IKK inhibitory properties including, but not limited to: SPC839 (Signal Pharmaceutical Inc.), Anilino-Pyrimidine Derivative (Signal Pharmaceutical Inc.), PS1145 (Millennium Pharmaceutical Inc.), BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute), SC-514*(Smithkilne Beecham Corp.). Amino-imidazolecarboxamide derivative (Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives (AstraZeneca), Diarylpybidine derivative (Bayer X Pyridooxazinone derivative (Bayer), Indolecarboxamide derivative (Aventis Pharma), Benzoimidazole carboxamide derivative (Aventis Pharma), Pyrazolo[4,3-c]quinoline derivative (Pharmacia Corporation), Imidazolylquinoline-carbxaldehyde semicarbazide derivative (Tulark Inc.), Pyridyl Cyanoguanidine derivate (Leo Pharma), I□B Kinase Inhibitor Peptide (CalBiochem), IKK-2 Inhibitor IV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (CalBiochem), IKK Inhibitor II (Wedelolactone (CalBiochem), IKK Inhibitor VII (CalBiochem), IKK-2 Inhibitor V (N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide IMD-0354, CalBiochem), IKK-2 Inhibitor VI (5-Phenyl-2-ureido)thiophene-3-carboxamide, CalBiochem), IKK-2 Inhibitor VIII (ACHP 2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxyphenyl)-(4-piperidinyl)-3-pyridinecarbonitrile, CalBiochem). ii) In a certain embodiment, the group of IKK inhibitors may additionally include compounds discovered to have IKK inhibitory activity, in accordance, and previously identified as anti-tumor agents, including, but not limited to PS1145 (Millennium Pharmaceutical Inc.), BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute).

Suitable WST-1r and the at least one of the valid substitutes of the WST-1r, as noted herein above, include, but are not limited to (1) the combination of at least one WST with at least one IEA. Examples as, but not limited to: WST-1+mPMS, WST-3+mPMS, WST-4+mPMS WST-5+mPMS, WST-9+mPMS, WST-10+mPMS, WST-11+mPMS, MSN+mPMS XTT+mMS, WST-1+coenzyme Q1, WST-3+coenzyme Q1, WST-4+coenzyme Q1 WST-5+coenzyme Q1, WST-9+coenzyme Q1, WST-10+coenzyme Q1, WST-11+coenzyme Q1, MSN+coenzyme Q1 XTT+coenzyme Q1; (2) at least one of the WST, such as, with no limitation, WST-3; (3) at least one IEA, such as, with no limitation, mPMS and coenzyme Q1 at optimized concentration in a pharmaceutically acceptable medium.

In a specific embodiment, the preferred order of treatment is to administer the pUC19 DNA transfection or its valid substitutes, at least one IKK inhibitor and WST-1r or at least one of the valid substitutes of WST-1r concurrently and/or sequentially in any type of order. However, the pUC19 DNA transfection or IFN treatment, or siRNA transfection or its other valid substitutes, at least one IKK inhibitor and the WST-1r or the at least one valid substitutes of WST-1r may be administered to the cells or patient concurrently or sequentially. In other words, the pUC19 DNA transfection may be treated first, the at least one IKK inhibitor may be administered first, the WST-1r or the at least one substitute of WST-1r may be administered first, or the pUC19 DNA transfection, the at least one IKK inhibitor and the at least one substitute of WST-1r may be administered at the same time. Additionally, when the pUC19 DNA transfection is replaced by siRNA transfection, IFN administration, or small molecule targeting the target genes as described in this description above, in combination with at least one IKK inhibitor and WST-1r or at least one valid substitute of WST-1r is used, the compounds may be administered in any order.

Cancers that may be treated using the present combinatorial protocol are carcinomas and sarcomas, lymphomars and leukemia include, but are not limited to those cancers described herein above in paragraph [00164]. However, the suitable cancer cells and tumors that may be more susceptible to this treatment are those with aberrant NF-κB activities.

The present description also provides additional methods for inducing cancer cell death and suppressing tumor in cancer patients. In accordance, it has been discovered that the combination of a flavonoid, apigenin, or its valid substitutes, or an IKK inhibitor at effective concentration with the WST-1r or the valid substitutes at effective concentration for synergistic induction of cancer cell death. Accordingly, the present description provides a pharmaceutical composition and protocol for the treatment of cancer in a patient in need with effective dose comprising of at least one flavonoid, preferably, apigenin, or its valid substitutes, or an IKK inhibitor with WST-1r or at least one of the valid substitutes of the WST-1r in a pharmaceutical acceptable medium.

A removal of the treatment is required for all the above and following embodiments to induce programmed cell death of the treated cells by this method.

Suitable flavonoids include, but not limited to, apigenin, the flavonoids, and valid substitutes of apigenin as described above in paragraph [00114 and 00115] in pharmaceutically acceptable medium.

The valid substitutes of apigenin are selected from the groups comprising The second compound and the valid substitutes of apigenin is selected from the groups comprising (1) At least one flavones, include., but not limited to nature existed flavones, such as: Luteolin, Tangeritin, Chrysin, 6-hydroxyflavone, Baicalein, Scutellarein, Wogonin and synthetic flavones, such as: Diosmin, Flavoxate, additional subgroups of flavones: flavonols, flavannones, flacanonols, catechins, isoflavones in paragraph [00114]; or (2) at least one from other subgroups of Flavonoid or Bioflavonoids and their isoforms including naturally existed, artificial modified isoforms and synthetic compounds including, but not limited to flavonoids, derived from 2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone) structure (examples: quercetin, rutin); isoflavonoids, derived from 3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure; neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-1,2-benzopyrone) structure, and flavanoids as a non-ketonepolyhydroxy polyphenol compounds as described above in paragraph [00114 and 00115]; or (3) At least one HIF inhibitors and/or inhibition of cellular responses to hypoxia including, but not limited to: 2,2-dimethybenzopyran compounds, chetomin, 2-methoxyestradiol (2ME2), PX-478, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), EZN-2968, camptothecins, NSC 644221, 3-(5′-hydroxymethyl-2-furyl)-1-benzylindazole (YC-1), rapamycin, and decoy oligonucleotides against HIF-1 RX-0047; as described above in paragraph [00116] in effective doses and in pharmaceutically acceptable medium.

Suitable IKK inhibitors are as listed above and following embodiments include any compound which exhibits IKK inhibitory activity in pharmaceutically acceptable medium. The at least one IKK inhibitor may be selected from compounds of the group consisting of, without limitation, i) compounds previously established to exhibit IKK inhibitory properties including, but not limited to: SPC839 (Signal Pharmaceutical Inc.), Anilino-Pyrimidine Derivative (Signal Pharmaceutical Inc.), PS1145 (Millennium Pharmaceutical Inc.). BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute), SC-514*(Smithkilne Beecham Corp.), Amino-imidazolecarboxamide derivative (Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives (AstraZeneca), Diarylpybidine derivative (Bayer), Pyridooxazinone derivative (Bayer), Indolecarboxamide derivative (Aventis Pharma), Benzoimidazole carboxamide derivative (Aventis Pharma), Pyrazolo[4,3-c] quinoline derivative (Pharmacia Corporation), Imidazolylquinoline-carbxaldehyde semicarbazide derivative (Tulark Inc.), Pyridyl Cyanoguanidine derivate (Leo Pharma), IκB Kinase Inhibitor Peptide (CalBiochem), IKK-2 Inhibitor IV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (CalBiochem), IKK Inhibitor H (Wedelolactone (CalBiochem), IKK Inhibitor VII (CalBiochem), IKK-2 Inhibitor V (N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide IMD-0354, CalBiochem), IKK-2 Inhibitor VI (5-Phenyl-2-ureido)thiophene-3-carboxamide, CalBiochem), IKK-2 Inhibitor VIII (ACHP 2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxyphenyl)-4-(4-piperidinyl)-3-pyridinecarbonitrile, CalBiochem). ii) In a certain embodiment, the group of IKK inhibitors may additionally include compounds discovered to have IKK inhibitory activity, in accordance, and previously identified as anti-tumor agents, including, but not limited to PS1145 (Millennium Pharmaceutical Inc.), BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb Pharmaceutical Research Institute).

Suitable WST-1r and the at least one of the valid substitutes of the WST-1r, as noted herein above, include, but are not limited to WST-1r and each of the individual components, the WST-1c anf mPMS, that are comprises the WST-1r, the valid substitutes for WST and that for IEA of the WST-1r and all possible combination among these valid substitutes of WST-1 and mPMS or the combination of these valid substitutes and the individual component of the WST-1r, the WST and IEA as described above in paragraph [00171], at optimized concentrations in pharmaceutically acceptable medium.

The effective concentration of apigenin that were used may vary depending on cell type. For all the above and following embodiments, the effective concentration of WST-1r and the valid substitutes may vary depending on the individual composition and the effective concentration of each of the composition may or may not be the same concentration as that in the Cell Proliferation WST-1 reagent and may vary from each of the compositions and their valid substitutes and between in vitro and in vivo usage.

In a specific embodiment, the administration of the WST-1r or at least one valid substitutes of WST-1r, the apigenin, the flavonoid or at least one of the valid substitutes of apigenin or the at least one IKK inhibitor can be in any type of order. Specifically, the WST-1r or at least one valid substitutes of WST-1r, and the apigenin or at least one of the valid substitutes of apigenin or the at least one IKK inhibitor may be administered to the cells or patient concurrently or sequentially. In other words, the apigenin or at least one of the valid substitutes of apigenin or the at least one IKK inhibitor may be administered first, the WST-1r or the at least one substitute of WST-1r may be administered first, or the WST-1r or at least one valid substitutes of WST-1r, and the apigenin or at least one of the valid substitutes of apigenin or the at least one IKK inhibitor may be administered at the same time. The preferred order of the treatment is to administer the WST-1r or the valid substitutes of WST-1r and the apigenin or the valid substitutes of apigenin or at least one IKK inhibitor simultaneously and then, after removal of the WST-1r, add apigenin or IKK inhibitor again and keep in contact with cells for another 24 hours.

In a particular embodiment, the in vitro treatment of WST-1r is in contact with cells for at least 15 minutes or longer. The preferred time is between 30 mm to 4 hours. The more preferred time is between 2-4 hours. A removal of the WST-1r or its valid substitute's from treatment is required for all the above and following embodiments to induce programmed cell death of the treated cells by this method thereof.

Moreover, the present description provides a method for the treatment of cancer by administering to a patient, in need thereof, a therapeutically effective dose of at least one of the WST-1r or its valid substitutes and apigenin or at least one of its valid substitutes as described above in pharmaceutical acceptable medium.

Also, the present description provides a method for the treatment of cancer by administering to a patient, in need thereof, a therapeutically effective dose of at least one of the WST-1r or its valid substitutes and at least one IKK inhibitor mentioned above in pharmaceutical acceptable medium.

Cancers that may be treated using the combinatorial protocol with WST-1r or its valid substitutes in combination with apigenin include, bid are not limited to those carcinomas and sarcomas that may be treated using the present protocol include, but are not limited to: cancers of the prostate, colorectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin (including melanoma and basal carcinoma), mesothelial lining, esophagus, breast muscle, connective tissue, lung (including small-cell lung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid, kidney, or bone; glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma, sarcoma, choriocarcinoma, cutaneous basocellular carcinoma, lymphoma, leukemia and testicular seminoma, soft tissue sacoma.

5. Other Compositions and Methods for Enhance and Synergize the Treatment Of Cancer

The present description provides additional methods for synergistic inhibition of NF-κB activity in cancer cells. In accordance, it has also been discovered that the pUC19 DNA transfection may also synergize the inhibition of NF-κB activity in cancer cells when both IKK1-KA and IKK2-KA kinase dead dominant negative vector were used simultaneously. This inhibitory effect can be further enhanced by the combination of additional treatment of WST-1r or at least one of the valid substitutes for WST-1r.

Accordingly, pUC19 DNA trasnfection may be substituted by treating the cells or a mammal with (i) administering a suitable dose of at least one IFN, or (ii) transfection of at least one specific siRNA or shRNA targeting at least one of the target transcripts as described previously in this specification, or (iii) small molecule inhibitors that targets at least one of the target genes products as described previously in this specification , or (iv) antibodies and peptide inhibitors targeting at least one of the target genes products as described previously in this specification, (v) anti-sense RNA targeting at least one of the target transcripts as described previously in this specification, (vi) anti-sense oligo targeting at least one of the target gene's transcripts as described previously in this specification in combination with the treatment of at least one IKK inhibitors that can inhibit both IKK1 and IKK2 kinase activities.

The at least one IFN may be selected from the subfamily of IFN including, but not limited to: IFNα A, IFNα B, IFNα C, IFNα D, IFNα F, IFNα G, IFNα H, IFNα I, IFNα J, IFNα K, IFNα 4b, IFNα WA, IFNβ, IFNγ or IL-6.

The transcripts, and proteins as the targets of the siRNA, shRNA, small molecule inhibitor, peptide inhibitor, antibody, anti-sense RNA, anti-sense oligo, and antibody are, but not limited to, (1) Homo sapiens transient receptor potential cation channel, subfamily C, member 6 (TRPC6, SEQ ID 2, 6), (2) Homo sapiens SH3 and FX domains 2B (SH3PXD2B, SeQ ID #3, #7), (3) Homo sapiens membrane associated guanylate kinase, WW and PDZ domain containing 3 (MAGIKK, SeQ ID #4, #8), (4) the Homo sapiens transmembrane protein 182 (TMEM 182, SeQ ID #5, #9) and (5) the C6orf108 (Seq ID #14, #15).

Suitable WST-1r and the at least one of the valid substitutes of the WST-1r, as noted herein above, include, but are not limited to WST-1r and each of the individual tetrazolium components that are comprises the WST-1r, the valid substitutes of each component of the WST-1r and any type of combination among these valid substitutes or the combination among these valid substitutes and the individual component of the WST-1 and mPMS.

The at least one IKK inhibitor may be selected from compounds of the group consisting of: i) compounds previously established to exhibit IKK inhibitory properties including, but not limited to: SPC839 (Signal Pharmaceutical Inc.), Anilino-Pyrimidine Derivative(Signal Pharmaceutical Inc.), PS1145 (Millennium Pharmaceutical Inc.), BMS-345541*(Bristol-Myers Squibb Pharmaceutical Research Institute), SC-514*(Smithkilne Beecham Corp.), Amino-imidazolecarboxamide derivative (Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives (AstraZeneca), Diarylpybidine derivative (Bayer), Pyridooxazinone derivative (Bayer), Indolecarboxamide derivative (Aventis Pharma), Benzoimidazole carboxamide derivative (Aventis Pharma), Pyrazolo[4,3-c]quinoline derivative (Pharmacia Corporation), Imidazolylquinoline-carbxaldehyde semicarbazide derivative (Tulark Inc.), Pyridyl Cyanoguanidine derivate (Leo Pharma), IkB Kinase Inhibitor Peptide (CalBiochem), IKK-2 Inhibitor IV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (CalBiochem), IKK Inhibitor II, Wedelolactone (CalBiochem), IKK Inhibitor VII K Inhibitor VII (CalBiochem), IKK-2 inhibitor V N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide IMD-0354 (CalBiochem), IKK-2 Inhibitor VI (5-Phenyl-2-ureido)thiophene-3-carboxamide (CalBiochem), IKK-2 Inhibitor VIII ACHP 2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxyphenyl)-4-(4-piperidinyl)-3-pyridinecarbonitrile (CalBiochem). In a certain embodiment, the group of IKK inhibitors may additionally include compounds discovered to have IKK inhibitory activity, in accordance, and previously identified as anti-tumor agents, including, but not limited to PS1145 (Millennium Pharmaceutical Inc.), BMS-345541*(Bristol-Myers Squibb Pharmaceutical Research Institute). The preferred IKK inhibitors are the IKK inhibitors that can inhibit both IKK1 and IKK2 kinase activities.

The present description provides additional medical use for inducing cancer cell death and tumor suppression, in accordance, it has been discovered that the combination of a GSK3β inhibitor with a CK2 inhibitor in combination with WST-1r or at least one of the valid substitutes for WST-1r act synergistically to suppress tumor growth. Accordingly, the present description provides a pharmaceutical composition for the treatment of cancer in a subset of cancer cells and/or in a patient comprising at least one GSK30 inhibitor, at least one CK2 inhibitor and WST-1r or the at least one of the valid substitutes for WST-1 in a pharmaceutically acceptable carrier. Also provided is a method for treating cancer in a patient by administering an effective amount of at least one GSK3β inhibitor in combination with at least one CK2 inhibitor. Suitable GSK3P inhibitors include any compound which exhibits GSK30 inhibitory activity, for example, LiCl. Suitable CK2 inhibitors, include, but are not limited to: Apigenin.

The at least one CK2 inhibitor may be selected from compounds of the group comprising, but not limited to: TBB, TBBz, emodin, CK2 inhibitor III (sigma).

Suitable WST-1r and the at least one of the valid substitutes of the WST-1r, as noted herein above, include, but are not limited to WST-1r and each of the individual components that comprises the WST-1r, the valid substitutes of each component of the WST-1r and any type of combination among these valid substitutes or the combination among these valid substitutes and the individual component of the WST-1r.

In a specific embodiment, the at least one GSK3P inhibitor and at least one CK2 inhibitor may be administered to the cancer cells or patient concurrently or sequentially. In other words, the at least one GSK3β inhibitor may be administered first, the at least one CK2 inhibitor may be administered first, or the at least one GSK3β inhibitor and the at least one CK2 inhibitor may be administered at the same time. Additionally, when more than one GSK3β inhibitor and/or CK2 inhibitor are used, the compounds may be administered in any order.

Cancer cells that may be treated using the present combinatorial protocol include, but are not limited to UM-SCC-6 cells. Cancers that may be treated using the present combinational protocol include, but are not limited to, those cancers described herein.

The present description provides additional medical use for enhancing or synergizing the efficacy effects of chemotherapy drugs for the treatment of cancer. In accordance, it has also been discovered that the Puc19 DNA transfection also synergizes suppression of tumor growth and promotes cancer cell death. Accordingly, the present description provides a pharmaceutical composition for the treatment of cancer in a patient comprising puc19 DNA transfection or at least one of its valid substitutes and at least one chemotherapeutic agent. This induction of cancer cell death effect may be further enhanced by additional combination with WST-1r or at least one of the valid substitutes of WST-1r in a pharmaceutically acceptable carrier. Also provided is a method for treating cancer cells or cancer in a patient by administering an effective dose of at least one DNA transfection or at least one of the valid substitutes for DNA transfection in combination with at least one chemotherapeutic agent In a preferred embodiment, the preferred DNA for transfection is pUC19 DNA cloning vector as described previous in this application (Sequence #1).

The at least one valid substitute for the pUC19 DNA transfection may include, but not limited to, (i) administering a suitable dose of at least one IFN, or (ii) transfection of at least one specific siRNA targeting at least one of the target transcripts as described previously in this specification, or (iii) at least one chemical compounds or small molecule inhibitors that targets at least one of the target genes and/or its gene products as described previously in this specification, or (iv) at lease one antibody targeting at least one of the target genes products as described previously in this specification, or (v) anti-sense RNA targeting at least one of the target transcripts as described previously in this specification, (vi) shRNA targeting at least one of the target transcripts as described previously in this specification, (vii) anti-sense oligo targeting at least one of the target transcripts as described previously in this specification, (viii) A dominant negative DNA vector targeting at least one of the target genes as described previously in this specification, (ix) peptides targeting at least one of the target genes products as described previously in this specification.

Suitable IFN may be selected from any IFN subfamily members, which include, but not limited to, IFNα A, IFNα B, IFNα C, IFNα D, IFNα F, IFNα G, IFNα H, IFNα I, IFNα J, IFNα K, IFNα 4b, WA, IFNβ, IFNγ and Interlukine-6 (IL-6). In a preferred embodiment, the preferred IFN are subfamily members of IFNα, IFNβ. The effective concentration of IFN is 10 unit/ml or lower for each IFN.

The target genes to be targeted by the at least one chemical compounds or small molecule inhibitors, at least one specific siRNA, shRNA, anti-sense RNA, anti-sense oligo, dominant negative DNA vector, at least one peptide, at lease one antibody, at least one inhibitor are, but not limited to, (1) TRPC6, (SEQ ID #2, #6), (2) SH3PXD2B, (SEQ ID #3, #7), (3) MAGIKK, (SEQ ID #4, #8), (4) TMEM 182, (SEQ ID #5, #9), and (5) C6orf108 (Seq ID #14, #15).

The gene products include, but not limited to, the nucleotide sequence of the transcripts from the gene and amino acid sequence of the protein that derived from these genes.

The siRNA and or shRNA sequences and the targets of the siRNA sequences may also include the nucleotide sequence that mapped to the human genomic sequences that flanking the genes as listed in the attached file “NCBI Blast-pUC19-Human-Transcripts and genome (2686 letters)” and “NCBI Blast_siRNA2 Nucleotide sequence (24 letters)”.

Accordingly, Suitable siRNAs include siRNA 1 (SEQ ID #10), siRNA 2 (SEQ ID #11), and siRNA 3 (SEQ ID #12) as described previous in this specification and all the potential siRNAs that may be derived from pUC19 DNA sequence that mapped to human genome and/or transcripts in short pieces (10-100 bp and more). These nucleotide sequences and their corresponding genes are listed in the attached file “NCBI Blast-pUC19-Human-Transcripts and genome (2686 letters)” and “NCBI Blast_siRNA2 Nucleotide sequence (24 letters)”. As in general, these siRNA sequences can be vary up to 40% from the exact sequences of the gene. Additionally, the function of these siRNAs can be substituted by any of the siRNA and/or shRNA that mapped to other part sequences of the corresponding target gene, small molecule inhibitors, peptide inhibitors, antibodies, anti-sense RNAs, anti-sense oligos and dominant negative DNA vectors that can effectively target the gene products as targets, which are the target of the siRNAs as described above in this paragraph and are include, but not limited to, (1) TRPC6, (SEQ ID #2, #6), (2) SH3PXD2B, (SEQ ID #3, #7), (3) MAGIKK, (SEQ ID #4, #8), (4) TMEM 182, (SEQ ID #5, #9), and (5) C6orf108 (Seq ID #14, #15).

The WST-1r or at least one of the valid substitutes of WST-11, as noted herein above, include, but are not limited to WST-1 rand each of the individual components that are comprises the WST-1r, the valid substitutes of each component of the WST-1r and any type of combination among these valid substitutes or the combination among these valid substitutes and the individual component of the WST-1r.

Suitable chemotherapeutic agents include, but are not limited to: paclitaxel (Taxol™), cisplatin, docetaxol, carboplatin, vincristine, vinblastine, methotrexate, cyclophosphamide, CPT-11, 5-fluorouracil (5-FU), gemcitabine, estramustine, carmustine, adriamycin (doxorubicin), etoposide, arsenic trioxide, irinotecan, and epothilone derivatives. The preferred chemotherapeutic agents are paclitaxel (Taxol™), cisplatin, 5-fluorouracil (5-FU), and

In a specific embodiment, the preferred order is to transfect the pUC19 DNA or at least one of its valid substitutes first and, then, administering the chemotherapy drugs after the transfection of pUC19DNA, However, the pUC19 DNA transfection or at least one of its valid substitutes and administering the chemotherapy drugs may be administered to the cancer cells or patient concurrently or sequentially. In other words, the pUC19 DNA transfection may be administered first; the chemotherapy drugs may be administered first.

Cancers that may be treated using the present combinatorial protocol include, but are not limited to those carcinomas and sarcomas set forth herein above.

Combined Treatment of Apigenin and Stattic Synergistic Inhibition of Ca127 Cell Survival and Induced Cell Death.

In addition to NF-κB, Signal transducer and activator of transcription (Stat) is another family of transcription factors. They mediate extra cellular signals stimulated by cytokines and growth factors, translocation to the cell nucleus where they act as transcription activators. These proteins mediate the expression of a variety of genes in response to cell stimuli, and thus play a key role in many cellular processes such as cell growth and apoptosis, Stat, such as STAT3, play an important role in cancer cells survival and proliferation. However, Stat Inhibitors or IKK inhibitors alone showed little inhibiting effect on cancer cell survival. Evidence showed that these two transcription factors interact with each other and to functionally cooperate with each other. In addition, NF-κB and STAT binding sites linked together to form promoter modules. Combination of Stattic, a Stat inhibitor with either IKK inhibitor or apigenin results in synergetic induction of cell death. This combination provides a method of treating cancer.

The present invention provides additional methods for inducing cancer cell death and tumor suppression. In accordance with the present invention, it has been discovered that the combination of a IKK inhibitor or a CK2 inhibitor in combination with Stat inhibitor, stattic, or at least one of the valid substitutes for stattic act synergistically to induce cancer cell death and to suppress tumor growth. Accordingly, the present invention provides a pharmaceutical composition for the treatment of cancer in a subset of cancer cells and/or in a patient comprising at least one IKK inhibitor or al least one CK2 inhibitor and stattic or the at least one of the valid substitutes for stattic in a pharmaceutically acceptable carrier. Also provided is a method for treating cancer in a patient by administering an effective amount of at least one IKK inhibitor or at least one CK2 inhibitor in combination with stattic or valid substitutes. Suitable IKK inhibitors are as listed above. Suitable CK2 inhibitors, include, but are not limited to: Apigenin. Suitable Stat inhibitors are the inhibitors that inhibit stat phosphorylation, activation and nuclear translocation, include, but not limited to stattic. The administration of the IKK inhibitors or the CK2 inhibitors and the stattic may be administered in any order. The preferred order is to administrate the inhibitors concurrently.

Advantages

From the description above, a number of advantages of the embodiments of this cancer treatment protocol and composition become evident:

This combination treatment targeting the tPMET and the HIF or cell responses to hypoxia is a synergistic combination strategy that block cancer cell respiration through the tPMET at cell surface while inhibit cancer cell capability of tolerating hypoxia. This combination did not inhibit cancer cell growth, but induced synergistic cancer specific cell death. This combination composition and method represent a new concept and principle tor a new avenue of cancer treatment strategy for a synergistic cancer specific treatment and anti-cancer drug development.

The chemical structure of WST-3 represents a model of a class of compounds that is capable of interfering the tPMET and restricts its activity on cell surface without affecting the mitochondrial in the normal cells. As cancer cells rely on cell surface oxygen consumption, the WST-3 represents the model of compounds that selectively affect cancer cells only.

The use of WST-1r also represents a novel strategy that incorporate cellular response to the treatment into the treatment protocol by inducing cancer cell tPMET followed by withdraw to induce cancer cells death.

This combination treatment is different from conventional chemotherapy that inhibits cancer cell growth, instead, it directly induce cancer cell death, which made it a more efficient cancer treatment by selectively killing cancer cells.

In summary this present invention provides a new concept of combinational treatment strategy for anticancer drug development This combination treatment will selectively block the cell surface respiration of cancer cell while inhibiting their capability to response to hypoxia therefore, to inhibit cancer cell respiration and hence the energy metabolism from two direction to obtain synergistic inducible cancer cell death. In addition, these treatments utilize non cytotoxic compounds result in synergistic cancer specific cell death, which provides a new avenue for anti-cancer drug development and for cancer treatment.

Although the description above contains much specificity, these should not be construed as limiting the scope of the embodiments but as merely providing illustrations of some of the presently preferred embodiments. For example, the WST-3 and the apigenin each represents classes of chemical compounds with similar function. Also the combination of WST-3 and apigenin represents a new strategy and a new avenue of cancer drug development by targeting tPMET in combination with inhibition of cellular responses to hypoxia and some other related process.

Thus the scope of the embodiments should be determined by the appended claims and their legal equivalents, rather than by the examples given.

V. Administration of Pharmaceutical Compositions and Compounds

The pharmaceutical compositions can be administered by any suitable route, for example, by injection, by intra vaneus infusion, by intra artery infusion, by oral, pulmonary, nasal, transdermal or other methods of administration. In general, pharmaceutical compositions of the present specification comprise, among other things, pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions can include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol). The compositions can be incorporated into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of components of a pharmaceutical compositions. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by reference. The pharmaceutical compositions can be prepared, for example, in liquid form, or can be in dried powder form (e.g., lyophilized). Particular methods of administering pharmaceutical compositions are described hereinabove.

In yet another embodiment, the pharmaceutical compositions can be delivered in a controlled release system, such as using an intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration, In a particular embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. (1987) 14:201; Buchwald et al., Surgery (1980) 88:507; Saudek et al., N. Engl. J. Med. (1989) 321:574). In another embodiment, polymeric materials may be employed (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press: Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley: New York (1984): Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. (1983) 23:61; see also Levy et al., Science (1985) 228:190; During et al., Ann. Neurol. (1989) 25:351; Howard et al., J. Neurosurg. (1989) 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the target tissues of the animal, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, (1984) vol. 2, pp. 115-138). In particular, a controlled release device can be introduced into an animal in proximity of the site of inappropriate immune activation or a tumor. Other controlled release systems are discussed in the review by Langer (Science (1990) 249:1527-1533).

The conclusion that this is programmed cell death is formed by the observation that normal cells had no cytotoxic reaction and further, that a over 90% kill rate is more than substantial evidence of a significant find. Therein, because this specification touches a programmed cancer cell dead) pathway that was prior untouched, or in the alternative, that this invention may activates a known pathway or a novel unknown pathway in a manner not able to be duplicated by other inventions, the very sequence of events defined in this specification activates programmed cell death in the cancer cells and as such, presents a valid model for further study. In other words, through processes known to those of skill, the very core molecular event leading to the over 90% kill rate, can be explored because we have the working model to induce such events. Therein, the invention is also claimed as an important model for further research, study and pathway illumination/elucidation.

Although above and below I have shown specific experimentation and data, one of skill in the art of cancer preclinical and clinical protocol structure, execution and analysis will recognize upon reading this document, through variation of the dosages of the named components, the order in which they are applied and the time frames between applications, valid substitutions of the named components there are a myriad of variable applications which may result in the same or similar outcome. To the extent that these variables can be applied to any cancer in any mammal, the inventor notes than nothing contained within this document or any subsequent documentation provided by the inventor is intended to be limiting. The inventor also notes that this specification is intended to work alone, and reduce cytotoxic effects of traditional cancer therapy, such as chemotherapy and radiation, however, nothing herein is intended to limit the use of this specification to the extent that chemotherapeutic and radiation combination therapies can be utilized in combination with this specification. Further, that the use of chemotherapy and radiation therapy combinations, in conjunction with this specification, may reduce the cytotoxicity of the chemotherapy or radiation therapy because the dosages of the chemotherapy and radiation therapy can be reduced when used in combination with this specification. And finally, that the named specification may further sensitize cancer cells selectively over normal cells such that subsequent application of chemotherapy and radiation, as well as combination chemo/radiation therapies, will work more efficiently again, allowing for the reduction of chemotherapy and radiation and combination chemo/radiation dosages.

The foregoing description of the present specification provides illustration and description, but is not intended to be exhaustive or to limit the specifications to the precise one disclosed. Modifications and variations consistent with the above teachings may be acquired from practice of the specification. Thus, it is noted that the scope of the invention is defined by the claims and their equivalents.

The present specification will now be illustrated in more detail in the following examples. It is to be understood that these examples serve only to describe the specific embodiments of the present specification, but do not in any way intend to limit the scope of the claims. It is of further note to one of skill that unique sequence data has been provided in this application. To the extent that each of these new sequence data represent novel targets for the development of cancer therapeutics, nothing contained herein is intended to be limiting. Said targets are noted as potential targets for further development under this application using the above methods and other methods known to those of skill. Although not mentioned in this specification elsewhere, use of radiation as a distinct step, or other small molecule drugs, DNA, RNA, siRNA and all other methods for cancer therapy known to those of skill are noted as possible adjuvant to these protocols.

VI. EXAMPLES Example 1 Synergistic Inhibition of NF-KAPPAB Activity

Overview: Normally, NF-kappaB activity is measured by reporter assay, electronic gel mobility shift assay and more recently, DNA binding ELISA. However, all of these methods employ exogenous DNA oligo or constructs carrying consensus NF-kappaB response element sequences for measuring specific NF-KAPPAB DNA binding and transcriptional activity. Additionally, the NF-KAPPAB consensus response element is different from the real promoter sequences that also need complex interaction with multiple molecules and may introduce artificial effects.

Method: UM-SCC-6 cells were transfected with effectene (Qiagen) i) 20% dominant negative IKK1-KA (K44A) and 80% pUC19, ii) 20% dominant negative IKK2-KA (K44A) and 80% pUC19, iii) 20% dominant negative IKK1-KA (K44A), 20% dominant negative IKK2-KA (K44A) and 60% pUC19, iv) 20% pCDNA3 and 80% pUC19 as negative control, for 72 hours. At the end of transfection, cells were lysed with lysis solution from GeneSpectra kit (Panomics). The IκBα, p100, CSNK2B mRNA levels were measured with the GeneSpectra kit. The expression levels of each transcript from different transfections were normalized by their 18sRNA level as measured at the same time.

Previously, we observed partial inhibition of NF-KAPPAB reporter activity by ˜50% caused by cotransfection of kinase dead K44A-IKK1 or K44A-IKK2 into UM-SCC-6 cells and other head and neck squamars carcinoma cells. By measuring expression levels of endogenous NF-KAPPAB downstream gene, IκBα, p100 and CK2β, as an indicator of NF-KAPPAB activity, we observed little inhibitory effect on NF-KAPPAB activity from K44A-IKK1 transfected cells (˜20%) and no inhibitory effect from K44A-IKK2 transfected cells. In contrast, when inhibiting both IKK1 and IKK2 molecules by cotransfecting dominant negative K44A-IKK1 and K44A-IKK2 simultaneously, we observed ˜90% inhibition at all three target gene expression levels that we measured (FIGS. 2 A and B). These data showed synergistic inhibitory effect of combination of K44A-IKK1 and K44A-IKK2 on constitutive NF-KAPPAB activity in these cancer cells, suggesting potential interchangeable function between these two IKKs.

Example 2 Simultaneous Inhibition of IKK1 and IKK2 Also Lead to Cancer Cell Death

In addition to the inhibition of NF-KAPPAB activity, cell death associated with cotransfection of K44A-IKK1 and K44A-IKKβ into UM-SCC-6 cells (FIG. 3). 48 hours after tranfection, K44A-IKK1 and K44A-IKK2 co-transfected cells showed 85% reduction in cell number (FIG. 3 WST-1 no) and dramatic cell death (FIG. 3B). The data represents the average of 7 sets of duplicates. This result indicates that inhibition of NF-KAPPAB activity does lead to cancer cell death and that this can be reached only by inhibiting both IKK1 and IKK2 simultaneously. In addition, adding tetrazolium dye WST-1r further enhanced cancer cell death caused by double inhibition of IKKs (FIG. 3 WST-1-yes). Following inhibition of both IKK1 and IKK2 treating cells with WST-1r further enhance cell death (FIG. 3 WST-1-yes). In FIG. 3A about 80% reduction of cell number in K44A-IKK1 and K44A-IKK2 cotransfected cells and over 95% reduction when these cells were treated with WST-1 in addition to cotransfection of K44A-IKK1 and K44A-IKKβ. Data represents an average of 7 sets of duplicates. FIG. 3B shows cell death from double transfected cells, partial cell death from K44A-IKK1 or K44A-1KK2 single transfected cells and further enhanced cell death by adding WST-1r treatment to K44A-IKK1 and K44A-IKK2 double trasfected cells.

Example 3

WST-1 Promote HT1080 Human Sarcoma Cell Death by Combination with DNA Transfection and IKK Inhibitor Treatment.

Methods: HT1080 cells were cultured in 96 well plates and transfected with one of the pUC19, pCDNA3, IKK1-KA, IKK1-KA+PUC19, or pCDNA3+pUC19 DNA vectors for 24 hours followed with treatment of IKK Inhibitor IIII at 3-30 μM for another 24 hours and, then, treated with 10% WST-1 for 4 hours and cultured overnight before detection. The same treatments of cells were measured at 24, 48 and 96 hours after WST-1 treatment. Cell viability was measured by Cell Count Kit 8 (CCK8).

Data showed (1) significant IKK inhibitor III dose dependent induction of cell death from the cells that were transfected with any of the DNA vectors at 24, 48 and 96 hour after WST-1 treatment comparing to non transfected control cells, but no significant difference between pUC19 vector only from IKK1-KA vector (FIG. 4); (2) further induced cell death and decreased cell survival detected from the cells that were treated with WST-1 at 24, 48 and 96 hours after WST-1 treatment comparing to those with no WST-1 treated cells; (3) at 96 hours after the treatment of WST-1 all the non transfected cells grow back to the same amount as the untreated control, while partial recovery of the cells from no WST-1 treated, but transfected cells; and (4) at 96 hours after all the treatment, only the cells transfected and also treated with WST-1 remained died with no recovery, which indicated the combination of either IKK inhibitor and/or DNA transfection with the WST-1 treatment further synergize these cancer cells to 100% death. The difference in the absorption at 24 hour after WST-1 treatment were caused, in partial, by decreased response from the WST-1 treated cells to the CCK8 detection. This effect reduced in 48 hours after the removal of WST-1 treatment and diminished at 96 hours after the removal of WST-1 treatment Morphology examination of the cells found that at 24 hours after the WST-1 treatment majority of the 30 μM IIKK inhibitor III treated cells died after the treatment. However, the survival cells that were not treated with WST-1 grow back up again. Conversely, the deaths of all the cells that were transfected with DNA vectors and with the same IKK inhibitor III treatment and treated with WST-1 were 100%. These data demonstrate the effect of WST-1 enhances the IKK inhibitor III induce cancer cell death effect and promote cell dead) of these cells and that pUC19 vector also contribute to the combined effect of inducing cell death.

Example 4

IFN Substitute pUC19 Transfection to Enhance IKK Inhibitor III and WST-1 Effect

Overview: Our previous data suggest that DNA transfection plays a role in the triple combination treatment for synergistic cancer cell death. Moreover, Interferon (IFN) responses have been reported to be involved in transfection effects. We examined whether IFN can be a substitute for the DNA transfection effect for in vivo treatment.

Methods: HT1080 cells were cultured in % well plates and treated with IFN, IKK inhibitor III and WST-1 sequentially. Each set of the cells were treated with one of the IFN members at the concentration ranging from 2-100 units/ml for 24 hours followed by IKK inhibitor HI treatment at concentration of 3-30 μM for another 24 hours and, then, with WST-1 for 4 hours and cultured overnight before detection. Cell viability was measured by CCK8 kit at 24 and 48 hours after WST-1 treatment. Total of 15 IFNs were tested. They arewer IFNα A, IFNαB, IFNαC, IFNαD, IFNαF, IFNαG, IFNαH, IFNαI IFNαJ, IFNαK, IFNα4b, and IFNαWA, IFNβ, IFNγ and IL-6.

Representative data (FIG. 21) showed IFN dose dependent and IKK Inhibitor HI dependent decrease of cell growth and enhancement of cell death comparing to that without IFN treatment. Comparing to pUC19 DNA transfection, which synergized the inhibition of cell growth and promotes cell death, IFN reached 80-90% inhibitory effects caused by pUC19 DNA transfection when combined with 30 μM BMS345541 and WST-1 at 48 hours after WST-1 treatment.

Example 5 WST-1 Induces ROS Generation

Overview: WST-1 was first described by ishiyama et at in 1996 (Ishiyam M, et al Biol Pharm Bull 1996, 19:1515-20). It is a cell proliferation detection reagent manufactured by Roche. WST-1 is composed of tetrazolium salt WST-1 {4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedilsulfonate} and an electron coupling reagent diluted in phosphate buffered saline. WST-1 can be cleaved by mitochondrial succinate-tetrazolium-reductase system. This cleavage has been used as the basis of the measurement of live cell. However, WST-1 has been found impermeable to cell membrane and their reduction occurs at the cell surface or at the level of the plasma membrane via trans-plasma membrane electron transport (Berridge, M V et al, Biotechnol Annu Rev, 2005; 11:127052). Alternatively, WST-1 can be reduced by cell surface NAD(P)H-Oxidase (Berridge, M V, et al, Antioxid Redox Signal. 2000, 2231-42, Scaled, D J, et al, Biofactors, 2004, 20:199-206) In the present invention WST-1 has been found to synergize the inhibitory effect on cell growth and promote cancer cell death when it is used in combination with at least one of the DNA transfection, or IFN, or siRNA transfection and one of the IKK, or combination of CK2 and GSK3□ inhibitors. Theoretically, it has been proposed that the balance between JNK activation and NF-KAPPAB activity determines cell faith to death or a live. Prolonged JNK activation induces programmed cell death. Generation of ROS induces JNK activation while NF-KAPPAB activity leads to suppress ROS level (Luo, J L, et al, J. Clin. Invest, (2005) 115:2625-32, Shen, H M, et al. Free Radical Biology & Medicine 2006, 40:928-939). The presort invention has found drat WST-1 induces ROS production in these cancer cells and promotes cell death.

Methods: HT1080 cells were cultured in cover slices and transfected with pUC19 and treated with IKK inhibitor III in sequential. Following these treatment, the cells, thus, treated, were either labeled with CM-H2-DCFDA, a fluorescence dye that can labeling ROS in cells, and, then, treated with WST-1 for 30 minutes (FIG. 23A) or treated with WST-1 for 2 hours and then labeled with CM-H2-DCFDA (FIG. 23-B). The results were recorded by a digital camera with Spotlight software. Manuel exposure levels were used to maintain the same exposure level for comparison.

In both experiments, significant WST-1 induced ROS generation has been documented (FIGS. 23 A and B). In FIG. 23-A, we also observed IKK inhibitor III dose dependent labeled ROS from the cells that were transfected with pUC19, and treated with IKK, but with no exposure to WST-1. This may suggest that IKK Inhibitor III may also induce ROS generation.

Example 6 LiCl+Apigenin Induced Synergized Cancer Cell Death, WST-1 Enhance Further These Cell Death.

Overview: LiCl is known to inhibit GSK3β activity and Apigenin is a multi signal transducer that inhibits multiple signaling processes, including protein kinase 2 (CK2). The activity of both GSK3β and CK2 are known to enhance constitutive NF-κB activity. This test was intended to examine whether combination of LiCl and Apigenin can substitute DNA transfection for the synergistic inhibitory effect and induction of cancer cell dead).

Methods: UM-SCC-6 cells were cultured in 96 well plates and treated with LiCl (1, 3, 10, 30, 100 mM) and Apigenin (1, 3, 10, 30, and 100 μM) in different combination of their doses for 24 hours followed by WST-1 treatment Cell viability was measured with CCK8 kit.

Data showed that combination of LiCl and Apigenin dose dependent decrease of cell growth and increased cell death comparing to untreated control cells. 10 μM AP and 100 mM LiCl showed synergistic increase of cell death (FIG. 5A). The subsequent treatment of WST-1 further enhanced this inhibitory effect (FIG. 5B).

Example 7

pUC19 DNA Transfection Synergize Chemotherapeutic Drug Effect in UM-SCC6 Cells

UM-SCC-6 cells were transfected with pUC19 DNA, pCDNA3, pUC19+pCDNA3, IKK1-KA+pUC19, IKK2-KA+pUC19, and IKKI-KA+IKK2-KA+pUC19, IKKI-KA+pCDNA3, IKK2-KA+pCDNA3, or IKKI-KA+IKK2-KA+pCDNA3, for 48 hours and, treated with variable doses of 5-FU (FIG. 6A) or Cis-Platinum (FIG. 6B) for 96 or 72 hours respectively. Cell viabilities were measured in 72 and 96 hours respectively after drug treatment. Data showed that pUC19 transfected cells showed the strongest inhibitory effects on cell growth.

Example 8

pUC19 DNA Transfection Synergize Chemotherapeutic Drug Effect in HT1080 Cells

HT1080 Cells were transfected with pUC19 DNA, pCDNA3, pUC19+pCDNA3, IKK1-KA+pUC19, IKK2-KA+pUC19, and IKK1-KA4+KK2-KA+pUC19, IKK1-KA+pCDNA3, IKK2-KA+pCDNA3, or IKK1-KA+IKK2-KA+pCDNA3, for 48 hours before the treatment of chemotherapy drugs at various doses. Cell viability was measured in 72 and 96 hours after drug treatment.

Drug treatment: Cis-Platinum 30 ng/ml-3 μg/ml (FIG. 6D), Paclitaxel 1 nM-10 μM (FIG. 6C), 5-FU 50 nM-500 μM (data not shown). Doxorubicin 30 nM-3.3 μM (data not shown).

Variable enhancement and synergistic effects were shown by the transfection of these DNA vectors, pUC19 DNA alone transfection showed the strongest synergistic efficacy effect to this chemotherapy dings comparing to other DNA vectors tested. IC50 of the drugs were lowered approximately 10 fold when combining pUC19 DNA transfected cells to that of untransfected cells of drug treatment (IC50 of Cis-Platinum from HT1080 cells and UM-SCC-6 cells untransfected control 3 μg/ml, pUC19 transfected cells 0.3 μg/ml and 1μg/ml respectively; IC50 of 5-FU from untransfected UM-SCC6 cells was more than 1 mM, but 200 MM from pUC19 transfected cells; IC50 of texal from pUC19 transfected HT1080 cells was 20 nM, while untransfected did not show any response upto 100 nM.). Furthermore, at 96 hours after of the cis-Platinum or palitaxel treatment untransfected cells recovered and grown up while the cell death from DNA transfected cells, especially pUC19 vector alone transfected cells were irreversible, meaning they were 100% died. These data suggest that these chemotherapy drugs inhibit cancer cell growth, but may not kill these cells. The combination of transfection of pUC19 DNA promote cell to death.

Example 9

Combination Treatment of Apigenin with WST-1r Synergizes Induced Cancer Cell Death

Method: UM-SCC6, MDA-MB-231, Ca127, HT1080, T294, B6-5 and A431 cells were treated with 3% or 10% of WST-1r or 10, 30, or 100 μM apigenin or combination of variable concentrations of WST-1r with apigenin in parallel with untreated control cells and DMSO control for 4 hours, then, the treatments were removed and the cells, thus, heated were changed to normal growth medium and maintained in culture for another 24 hours. DMSO was used as vehicle control. Cell viabilities were measured by CCK8 Kit and normalized to % of untreated control calls.

Result: DMSO treated cells of every tested cell line showed same levels of cell viability as untreated control cells (data not shown).

A: UM-SCC6, MDA-MB-231, Ca127, HT1080, T294, B6-5 and A431 cells were treated with 3% of WST-1r or 100 μM apigenin or combination of 3% WST-1r with 100 μM apigenin in parallel with untreated control cells. Date showed that the combination of WST-1r and apigenin induced 75% to 95% cell death of all seven tested cancer cell fines comparing to untreated controls (FIG. 8A).

B-E: Data showed both WST-1r and apigenin dose dependent cell death and synergized cell death effect when combining 3% WST-1r with 100 μM apigenin or 10% WST-1r with 30 μM apigenin. B & C: WST-1r Dose-Response of MA-MB-231 cells (B) and A431 cells (C). The IC50 of WST-1r in the presence of 100 μM apigenin were 1% for both MDA-MB231 cells and A431 cells, while treatment of apigein alone showed little effect on cell survival D & E: apigenin Dose-Response of MDA-MB-231 cells and A431 cells. The apigenin IC50 in the presence of 10% WST-1r were 10 μM for both cell lines (FIG. 8 B-E).

Example 10

Comparison of cell responses to modified WST-1r and Apigenin combination treatment between cancer cells and non-cancer cells. UM-SCC6, Ca127 human head and neck cancer cell lines and primary cultured human bronchia keratinocytes (HEKa) as labeled were treated with 10% modified WST-1r (MPMS 20 μM, WST-1c 1 MM) with (WST-1R 10) or without (WST-1r 0) 10% modified WST-1r in combination with 0 (Apigenin 0), 30 (Apigenin 30) or 100 (Apigenin 100) μM Apigenin for 4 hours, then, changed to apigenin in the corresponding concentrations for another 24 hours. Data showed that each of the WST-1r and the Apigenin single agent treatment had little effect on cell viabilities (FIG. 9). Combination of WST-1r with 100 μM Apigenin resulted in synergistic cell death in both UM-SCC6 and Ca127 cancer cell lines, but not in paired non-cancer primary cultured HEKa cells (FIG. 9). These data demonstrated the cancer cell specificity of this combination treatment.

Example 11

Time Course and Dose Response of WST-1r and Dose-Response of Apigenin Involved in the Combination Treatment of WST-1r with Apigenin

Methods: Cal27 (A), HT1080 (B) and UM-SCC6 (C) cells were treated with variable concentration (1%, 3% and 10%) of WST-1r as indicated for 0.5, 1, 2, and 4 hours in combination with variable doses of apigenin (3, 10, 30 and 100 μM) as indicated and, then, treated with the same concentration of apigenin for another 24 hours. Cell viabilities were measured by CCK8 Kit and normalized as % of that of untreated control calls.

Results: Data showed WST-1r time and dose dependent and apigenin dose dependent cell death from all three tested cell lines (FIG. 10A-C). Synergetic induction of cell death (over 80%) occurred at 10% WST-1r treatment for 0.5 hour in combination with 100 μM apigeinin treated Cal27 and UM-SCC6 cells (FIG. 10A, C) and at 3% WST-1r treatment for 1 hour in combination with 100 μM apigeinin treated HT1080 cells (FIG. 10B). By increasing WST-1r treatment time, 80-90% cell death can be reached at 30 μM apigenin in combination with 4 hour 10% WST-1r treatment from HT1080 and UM-SCC6 cells (FIGS. 10B&C) and at 30 μM apigenin in combination with 1 hour 10% WST-1r treatment from Cal 27 cells (FIG. 10C). (Ap=apigenin; uM=μM)

Example 12

Effect of Combination Treatment with IKK Inhibitor and WST-1r on Melanoma Cell Lines

Method: SK-Mel-5 and T294 human melanoma cells were treated with WST-1r at 1% and 3% final concentration respectively as indicated for 4 hours, then, removed WST-1r by changing to normal growth medium and added IKK inhibitor III for another 24 hours. After 24 hours treatment with 3 μM and 10 μM IKK inhibitor III respectively as indicated, cells were changed to grow in normal growth medium for 48 hours before measuring cell viability by CCK8 Kit.

Result: Both SK-mel-5 cells (FIG. 11A), and T294 cells (FIG. 11B) showed WST-1r and IKK inhibitor BMS345541 dose dependent increase of cell deaths. Combination of 3% WST-1r and 10 μM BMS345541 further synergized the induction of cell (FIGS. 11A&B). Whereas, the non-cancer primary cultured human keratinocytes were resistant to this combination treatment (FIG. 11C).

Example 13 Effects of Treatment Order of WST-1r and IKK Inhibitor III (BMS345541) on Inducing Human Melanoma Cell Death

Method: T294 cells were treated with 3% of WST-1r in combination with 3 or 10 μM BMS345541 respectively indifferent order as indicated. Cell viabilities were measured by CCK8 Kit and normalized as % of that of untreated control calls. Control: Cells were either untreated or treated with BMS345541 only at the indicated doses for 24 hours and, then, changed to normal growth medium for another 24 hours before measuring cell viability. B→W: Cells treated with BMS345541 only at the indicated doses for 24 hours and, then, added WST-1r at 3% final concentration for 4 hours, then removing the treatment and changed to normal growth medium for another 24 hours before measuring cell viability. W+B: Cells treated with 3% WST-1r and BMS345541 at the indicated doses for 4 hours and, then, removing the treatment and changed to normal growth medium for another 24 hours before measuring cell viability. W+B→B: Cells treated with 3% WST-1r and BMS345541 at the indicated doses for 4 hours and, then, removing the treatment and added BMS345541 at the indicated doses in normal growth medium for another 24 hours before measuring cell viability. W→B: Cells treated with 3% WST-1r for 4 hours and, then, removing the treatment and added IKK BMS345541 at the indicated doses in normal growth medium for another 24 hours before measuring cell viability.

Result: Data showed that W→B and W+B→B treatment orders synergized induction of cell death (FIG. 12).

Example 14 WST-1r and Apigenin Combination Treatment Induced JNK Phosphrylation

Method: UM-SCC6 cells were treated with WST-1r and apigenin at the indicated doses for 4 hours, and then phosphorylated JNK and total JNK were measured in parallel with FACE Kit (Qiogen). The resulting data were normalized to total cell number measured by crystal violet staining. The phosphorylated JNK from each measurement were normalized to the ratio of phosphorylated JNK over total JNK values.

Result: Data showed WST-1r and apigenin dose dependent induction of phosphorylation of JNK in UM-SCC6 cells (FIG. 13). Combination of WST-1r and apigenin further increased JNK phosphorylation. The most significant increase of JNK phosphorylation from UM-SCC6 cells occurred at the treatment of 100 μM apigenin in combination with 3% or 10% WST-1r. This result supports the hypotheses that the combination of WST-1r with apigenin treatment induced JNK activation.

Example 15 Dose Response of ROS Generation After Combination Treatment of WST-1r and Apigenin and IKK Inhibitor III

Method: UM-SCC6 cells were labeled with 10 μM CM-H2-DCFDA for 15 minutes and then treated with WST-1r or CCK8 at the indicated amounts in combination with variable doses of apigenin (A) or IKK Inhibitor III (B) for 4 hours. Fluorescence at Ex485/Em535 were measured for detecting ROS generation that labeled by the CM-H2-DCFDA.

Result: Data showed WST-1r dose dependent induction of ROS generation (FIGS. 24A and B). On the other hand, CCK8 induced low and very limited level of ROS generation with no relation to the CCK8 treatment dose at 4 hours after the treatment. Apigenin alone showed no effect on ROS generation. However, combination of apigenin with 1% and 3% WST-1r did show apigenin dose dependent, limited, but, steady increase on ROS generation from thus treated cells when comparing to that of the corresponding doses of WST-1r only treated cells. (FIG. 24-A) Conversely, combination of 10% of WST-1r with apigenin resulted in decrease of ROS levels (FIG. 24-B). In addition, when combined with CCK8, apigenin also increase the ROS generation (FIG. 24-A). This effect is apigenin dose dependent.

Similarly, IKK inhibitor III alone and combination of WST-1r with IKK inhibitor III (FIG. 24-B) showed similar effect as apigenin did, where 5 μM IKK Inhibitor increased ROS levels while 10 μM IKK Inhibitor III decreased it (FIG. 24-B). However, IKK Inhibitor III had no combined effect with CCK8 on ROS levels.

Example 16 Time Course of ROS Generation After Combination Treatment of WST-1r and Apigenin and IKK Inhibitor III

Method: UM-SCC6 cells were labeled with 10 μM CM-H2-DCFDA for 15 minutes and then treated with WST-1r (B & D) or CCK8 (A & C) at the indicated amounts in combination with variable doses of apigenin (C & D) or IKK inhibitor III (A & B) for the time period from 15 minute up to 4 hours. At each time points as indicated, fluorescence at Ex485/Em535 were measured for detecting ROS generation that labeled by the CM-H2-DCFDA.

Result: Data showed that WST-1r induced ROS generation continued increase and lasted at least for more than 4 hours (FIGS. 7-B & D), whereas, CCK8 only induced low level and transience increase of ROS (FIGS. 7-A & C).

Example 17 CCK8-XTT-WST-1 Comparison

Comparison cell death inducing capability of CCK8 and XTT to WST-1r in combination with apigenin treatment

Method: HT1080 and UM-SCC6 cells were treated with 10% of WST-1r, CCK8 or XTT in combination with variable doses of apigenin for 4 hours and, then changed to normal growth medium for another 24 hours. Cell viability was measured with CCK8 kit.

Result: Data showed that CCK8 had no effect on cell death when comparing to control cells, while XTT showed intermediate induction of cell death effect comparing to WST-1r on both UM-SCC6 (FIG. 14B) and HT1080 cells (FIG. 14A). Apigenin IC50 of WST-1r and, XTT treated UM-SCC6 cells were 5 and 25 μM while apigenin only and CCK8+apigenin treatments did not reached IC50. Similar result from HT1080 cells as well.

Example 18 Effects of Other Tetrazolium Salts as Substitutives of WST-1r for Combination Treatment

Method: HT1080 and UM-SCC6 cells were treated with 1 mM WST-1, 0.4 mM WST-3, 0.5 mM WST-4, 0.5 mM WST-5 or 0.12 mM mPMS alone or each of the WST-3, WST-4, and WST-5 at the same concentration in combination with 0.12 mM mPMS (0.4 mM WST-3+0.12 mM mPMS, 0.5 mM WST-4+0.12 mM mPMS, 0.5 mM WST-5+0.12 mM mPMS) plus 10, 30 or 100 μM apigenin for 4 hours and, then changed to normal growth medium for another 24 hours. Cell viability was measured with CCK8 Kit.

Result: Data showed that WST-3 alone, WST-3+mPMS and WST-4+mPMS in combination with apigenin showed similar synergistic effect on inducing cell death that equivalent to that WST-1r does from both H11080 cells (FIG. 17A) and UM-SCC6 cells (FIG. 17B). WST-1, WST-4, and WST-5 alone showed no such effect (FIG. 17 A,B). WST-3+mPMS are more potent than WST-1r on cell death induction.

Example 19

mPMS Dose-Response

Method: A & B: HT1080 (FIG. 16A) and UM-SCC6 (FIG. 16B) cells were treated with variable concentrations of mPMS as indicated in combination with 1 mM WST-1c and 10, 30 or 100 μM apigenin for 4 hours and, then, changed to normal growth medium for another 24 hours. Cell viabilities were measured by CCK8 Kit. 1 mM WST-1 only, 0.12 mM mPMS only and 10% WST-1r were used as parallel control. AP 0: Untreated Control, AP 10: 10 μM Apigenin, AP 30: 30 μM Apigenin, AP 100: 100 μM Apigenin.

Result: Data showed mPMS and apigenin dose dependent cell death of both HT1080 and UM-scc6 cells (FIGS. 16A &B). mPMS IC50 of combination treatment of apigenin 100 μM and mPMS+WST-1 from HT1080 cells was 5 μM verses 60 μM from untreated control cells. mPMS IC50 of combination treatment of apigenin 100 μM and mPMS+WST-1 from UM-SCC6 cells was 30 μM verses 80 μM from untreated control cells.

Example 20

Differential Cellular Responses to mPNS Treatment

Non cancer human keratinocyte (HEKa), SK-Mel-5 human malonoma cell line (SK5), human head and neck cancer cell Cal27 line (Cal27) and UM-SCC6 line (SCC6) cells and human soft tissue sarcoma cell line HT1080 (HT1080) were treated with 30, 40 or 50 μM mPMS for 4 hours and then cultured in normal growth medium for another 24 hours. Cell viabilities were measured with CCK8 kit. Data showed mPMS dose dependent cell death and differential sensitivities to mPMS treatment from each of the cell lines (FIG. 17). Among those, the non cancer primary cultured HEKa cells showed the least sensitivity to mPMS treatment with IC50 50 μM, while the IC50 from Cal27, UM-SCC6 and HT1080 cells were 20, and 30 μM respectively.

Example 21

Effect of Combination Treatment of WST-3 with Apigenin on Induction of Cancer Cell Death

Method: UM-SCC6, HT1080, Cal27, SK-Mel-5, and HEKa cells were treated with 50 or 100 μM WST-3 or 10 or 30 μM apigenin alone, or combination of WST-3 and apigenin at different concentrations for 4 hours with untreated cells as control, then, changed to normal growth medium and remained culture in this medium for another 24 hours. After the 24 hours culture, cell viabilities were measured with CCK8 Kit. Data were normalized to % of untreated control cells.

Result: A: Summary of differential cell responses to WST-3, apigenin and combination treatments. Comparing to untreated cells (Ctrl) treatment of 50 μM WST-3 (WST-3) or 30 μM apigenin (Apigenin) alone showed no or limited effect of cell death to all tested cell line. Combination of WST-3 and apigenin (Apigenin+WST-3) resulted in synergistic cell death of SK-Mel-5, Cal27, UM-SCC6 and HT1080 all four tested human cancer cell lines, but limited cell death from non cancer human keratnocytes (FIG. 18A).

B & C: Comparison of Dose-Response of WST-3 and apigenin between non cancer HEKa and human melanoma cell line SK-Mel-5 cells. Data showed both WST-3 and apigenin induced and dose dependent but limited cell death from both HEKa cells (FIG. 18B) and SK-Mel-5 (FIG. 18C) cells. HEKa cells showed limited cell death in response to apigenin or WST-3 alone treatment. WST-3 IC50 of combination of 30 μM apigenin and WST-3 was 40 μM. Further increase WST-3 concentration showed no more cell death from HEKa cells (FIG. 18B). However, the SK-Mel-5 cells showed synergistic cell death response to combination treatment of 50 μM WST-3 and 30 μM apigenin. The WST-3 IC50 from this combination treatment of the SK-Mel-5 cells was 20 μM, one fold less than that from HEKa cells (FIG. 18C). The HEKa cells were much more resistant to this combination treatment. Similar results were also observed from other cancer cells.

Example 22

Effect of Substitution of WST-1r with WST-3+mPMS for Combination Treatment with Apigenin on Induction of Cell Death

Method: UM-SCC6, HT1080, Cal27, SK-Mel-5, and HEKa cells were treated with 0.1 mM WST-3 plus 30μM mPMS, or WST-3 only, or untreated control in combination with 10 or 30 μM apigenin for 4 hours and, then, changed to normal growth medium and remained culture in this medium for another 24 hours. After the 24 hours culture, cell viabilities were measured with CCK8 Kit. Data were normalized to % of untreated control cells.

Result: Data showed that over 90% induced cell death observed from thus treated Cal27, UM-SCC6, and SK-Mel-5 cells that were treated in combination of 0.1 mM WST-3. and 30 μM apigenin (FIG. 19C). Adding 30 μM mPMS to these treatments further synergize the cell death from Cal27 and UM-SCC6 cells (FIG. 19C). On the other hand, under this treatment condition, HEKa, primary cultured human keratinocytes, were relative resistant to this treatment. This difference in sensitivity to this combination treatment may provide a window tor differentiating targeting cancer cells and to control toxicity to normal cells.

Example 23

Enhancement of Taxel Efficacy Effects by Combination of Puc19 DNA Sequence Derived siRNA with Taxel

Method: HT1080 cells transfected with siRNAs that were derived from Puc19 DNA sequence and the siRNAs that targeting the corresponding genes that are the targets of the Puc19 derived siRNAs for 24 hours, then, treated with Taxel at 3, 10, 30, and 100 nM for 48 hours. After the 48 hours of Taxel treatment, cells in culture were changed to normal growth medium for 24 to 72 hours. Cell viability was monitored by CCK8 Kit every 24 hours. Data are normalized to % of untreated control cells. The siRNAs that used for this study includes siRNA#1, siRNA#2, siRHA#3, siRNA targeting TRPC6, SH3PXD2B. C6orf108, TTBK1, MAGI3, and TMEM182 as well as combination of siRNA#2+#3, and siRNA#1+#2°#3°#4+#5 (siRNAΣ1-5).

Result: Data showed represent the measurements of 72 hours after the treatment. The cell survival data showed Taxel dose dependent cell death and enhanced cell death by Puc19 trasnfection and majority of the siRNA trasfections (FIG. 22). The IC50 of taxel (Contrl IC50: 60 nM) was reduced more than 3 fold by Puc19 DNA trasnfection (IC50: 20 nM) and by the trasnfeciton of siRNAs targeting TRPC6 (IC50: 25 nM FIG. 22A), SH3PXD2B (IC50: 20 nM FIG. 22C), C6orf108 (IC50: 20 nM FIG. 22C), TTBK1 (IC50: 35 nM FIG. 22C), MAGI3 (IC50: 20 nM FIG. 22A), and TMEM182 (IC50: 20 nM FIG. 22B), as well as by the transfection of combination of siRNA#2+#3 (IC50: 25 nM FIG. 22A), B, and siRNA#1+#2+#3+#4+#5 (IC50:20 nM FIG. 22A-C). Over 2.5 fold IC50 decrease of taxel concentration were observed from siRNA#2 (IC50: 25 nM FIG. 22C), siRHA#3 (IC50: 25 nM FIG. 22B), and siRNA targeting SH3PXD2B (IC50: 25 nM FIG. 22C), C6orf108 (IC50: 20 nM FIG. 22C). These data demonstrated that the DAN sequences of Puc19 DNA vector code for some short functional sequences that can target and interrupt the expression levels of the corresponding genes and the cellular functions. At least the genes (TRPC6, SH3PXD2B, C6orf108, MAGI3, and TMEM182) that have been tested can be used as a target for anti-cancer drug design for enhancing the efficacy effect of chemotherapy drugs. The siRNAs targeting these corresponding genes that were identified may also be used as a tool to reach this goal. In addition, these combined treatments induce cancer cell death rather than simple inhibition of cell growth. 72 hours after treatment, the treated cells did not grow back. This feature adds to lasting effect of the treatment.

Example 24

Substitution of Puc 19 with siRNA Against TMEM 182 and MAGI3 for Puc19-IKK Inhbitor-WST-1r Triple Combination Treatment

Method: HT1080 cells were transfected with siRNA#3, or the siRNA targeting MAGI3, and TMEM182 that were derived from Puc19 DNA sequence and the siRNAs that targeting the corresponding genes that are the targets of the Puc19 derived siRNAs for 24 hours, then, treated with IKK inhibitor III for 24 hours followed by adding WST-1r for another 4 hour. After the 4 hours WST-1r treatment, cells in culture were changed to normal growth medium for 24 hours. Cell viability were monitored by CCK8 Kit every 24 hours. Data were normalized to % of untreated control cells. AllStar siRNA was used as negative siRNA trasnfection control. Puc19 DNA vector transfciton was used as positive control.

Data label: Untreated control: 0 Ctrl, WST-1r only: 10 Ctrl, pUC19 trasnfected cells: 0 p9, pUC19 transfected and WST-1r treated: 10 p9, AllStar negative contrl siRNA transfected: 0 AllStar, AllStar transfected and WST-1r treated: 10 AllStar, siRNA#3 transfected: 0 siRNA#3, siRNA#3 transfeted and WST-1r treated: 10 siRNA#3. siRNAMAGI3 transfected: 0 MAGI3, siRNA MAGI3 transfeted and WST-1r treated: 10 MAGI3, siRNATMEM182 transfected: 0 TMEM182, siRNATMEM182 transfeted and WST-1r treated: 10 TMEM182.

Result: Data showed IKK inhibitor BMS345541 dose dependent cell death and that siRNA targeting MAGI3 and TMEM182 synergize the ceil death (FIG. 20). At 15 μM BMS345541 incombinatoin with 10% WST-1r and either pUC19 or the siRNA trasnfeection resulted in synergistic induction of cell death. Again, these data showed that targeting TMEM182 and MAGI3 may enhance effect on cancer treatment. 

What is claimed:
 1. Biological molecules of Targets used for designing anticancer drugs are comprising but not limited for a) Polynucleotide sequences b) Polypeptide sequences which, when been inhibited in expression levels and/or inactivated functionally, sensitize cancer cells to chemotherapeutic drug treatments, hereby, be used as target for developing anticancer drugs.
 2. According to claim 1 Biological molecular targets for anticancer drug development wherein said polynucleotide sequences comprising i. a polynucleotide of (SEQ ID Nos: 2, 3, 4, 5, 14) ii. a polynucleotide encoding a polypeptide of (SEQ ID Nos: 6, 7, 8, 9, 15) iii. a polynucleotide which is a variant of (SEQ ID NoS: 2, 3, 4, 5, 14) iv. a polynucleotide which is an allelic variant of (SEQ ID NoS: 2, 3, 4, 5, 14) v. a polynucleotide which encodes a species homologue of the (SEQ ID NoS: 2, 3, 4, 5, 14)
 3. According to claim 1 Biological molecular targets for anticancer drug development wherein said polypeptide sequences comprising a. polypeptide of SEQ ID NoS:: 6, 7, 8, 9, 15 b. a polypeptide domain of SEQ ID NoS:: 6, 7, 8, 9, 15 c. a polypeptide epitope of SEQ ID NoS:: 6, 7, 8, 9, 15 d. a full length protein of SEQ ID NoS:: 6, 7, 8, 9, 15 e. a variant of SEQ ID NoS:: 6, 7, 8, 9, 15 f. an allelic variant of SEQ ID NoS:: 6, 7, 8, 9, 15 g. a species homologue of SEQ ID NoS:: 6, 7, 8, 9, 15
 4. According to claim 1, the anticancer treatment target that can be targeted by the molecules that are capable of interfering, affecting or inhibiting the expression levels and/or interfering, affecting or inhibiting the functional activities of the targeted molecules or inactivating the target molecules functionally, to sensitize cancer cells to chemotherapeutic drug treatments comprising, but do not limited to a) the nucleotide sequences, synthetic, not synthetic, modified or not modified nucleotide molecules that are capable of being complemented to at least one of the sequence of sequences #2, #3, #4, #5, #14; b) the nucleotide sequences, synthetic, not synthetic, modified or not modified nucleotide molecules that are capable of being complemented to at least one of the sequence of sequences #2, #3, #4, #5, #14 under strictly conditions; c) the biological molecules that are capable of binding to, or altering, or interfering with, or blocking, or inhibiting the expression levels and/or functional activities of at least one of the peptide sequence with the sequence # of #6, #7, #8, #9 and/or #15 comprising polypeptides, proteins, antibodies, and antibody fragments; d) Small molecule compounds that are capable of binding to, or altering or interfering with, or blocking, or inhibiting the expression levels and/or functional activities of at least one of the peptide sequence with the sequence # of #6, #7, #8, #9 and/or #15;
 5. According to claim 4

!

∘, means for binding to the molecules, or altering or interfering with, or blocking, or inhibiting the expression levels and/or functional activities of at least one of the of the said genes and gene products comprising a. synthetic or non synthetic polynucleotide, b. modified or not modified c. dominant negative DNA sequences, d. DNA oligos, modified or not modified e. PNAs f. siRNAs g. anti-sense RNAs h. shRNAs i. polypeptides, modified or not modified i. antibodies j. antibody fragments k. polypeptides l. organic or inorganic molecules m. small molecule antagonists
 6. synthetic polynucleotide further selected from the group of siRNA that were capable of altering, interfering, suppressing the expression levels and inhibiting/interfering the functional activities of the gene products of sequence comprising a. TPCR6 (SEQ ID NO:2), and/or b. SH3PXD2B(SEQ ID NO:3), and/or c. MAGI3(SEQ ID NO:4), and/or d. TMEM182(SEQ ID NO:5), and/or e. C6orf108(SEQ ID N0:14), and/or f. siRNA#1 (SEQ ID N0:10), and/or g. siRNA#2 (SEQ ID NO:11), and/or h. siRNA#3 (SEQ ID NO:12). 